Detection of autoantibodies against NR1

Abstract

This disclosure provides a system and method for detecting anti-NMDAR autoantibodies based on the strong affinity of multiple non-random anti-NR1 anti-NMDAR autoantibodies coupled to a solid support. This disclosure further provides a therapeutic method for anti-NMDAR pathology using the therapeutic anti-NMDAR antibody, ART5803. This disclosure further provides a method and system for screening and predicting potential responsiveness to ART5803 treatment.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 493,217, filed on 30 March 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The above XML copy, created on 26 March 2024, is named 62548-705_601_SL.xml and has a size of 68,612 bytes. [Background technology]

[0003] N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors known to be involved in various functions in the central nervous system. They play a crucial role in synaptic plasticity, long-term potentiation, and memory formation. NMDARs are activated upon binding of glycine and glutamate. Functional NMDARs are typically heterotetramers consisting of two NR1 subunits with either two of the four NR2 subunits or two NR3 subunits. The glycine-binding modules of the NR1 and NR3 subunits and the glutamate-binding module of the NR2 subunits allow for precise control of ion permeability under physiological conditions. Ca2+ 2+Overactivation of NMDARs, which causes an excessive influx of calcium, can lead to excitotoxicity known to occur in numerous neurological disorders, including Alzheimer's disease, Parkinson's disease, epilepsy, and Huntington's disease. On the other hand, decreased NMDAR function is known to be associated with neuropsychiatric disorders, particularly psychiatric disorders and schizophrenia. NMDARs are also associated with anti-NMDAR encephalitis, a rare autoimmune disease characterized by psychiatric and neurological symptoms such as memory loss, hallucinations, delusions, and seizures. This is caused by the production of immunoglobulin G (IgG) autoantibodies against the NR1 subunit of NMDARs in the blood and brain. Anti-NMDAR autoantibodies induce cross-linking and internalization of NMDARs. Decreased levels of cell surface NMDARs lead to decreased neuronal Ca2+ levels. 2+ This results in reduced inflow and decreased synaptic current, which is the cause of several neuropsychiatric and neurological symptoms observed in patients. [Overview of the project]

[0004] Currently, there are no established methods for screening and / or detecting anti-NMDAR autoantibodies from blood samples and other readily available patient samples. Typical detection methods using anti-NR1 antigens, such as enzyme-linked immunoassays (ELISA) or cell-based fluorescence-activated cell sorting (FACS), require high concentrations of anti-NMDAR autoantibodies in serum / blood and therefore lack sensitivity. Lumbar puncture would be impractical for diagnosing and studying NMDAR-related mental disorders. Furthermore, for diseases with lower concentrations of anti-NMDAR autoantibodies or weaker affinity anti-NMDAR autoantibodies, the levels of anti-NMDAR autoantibodies in cerebrospinal fluid (CSF) may still fall below the thresholds of these detection methods. Described herein are compositions, methods, and systems for detecting pathological anti-NMDAR autoantibodies in biological samples of individuals. The advantage of this detection method and system is that it uses multiple non-randomized NMDAR antigens coupled to a solid support in a unidirectional and density-controlled manner, thereby enabling stronger binding by two arms of anti-NMDAR autoantibodies to NMDAR antigens compared to conventional methods that use NMDAR antigens randomly coated on a solid support. The disclosed detection method and system can detect levels of IgG / IgA / IgM anti-NMDAR autoantibodies in patients with either lower concentrations and / or weaker affinity of anti-NMDAR autoantibodies. In addition, this detection system can be further used to predict therapeutic responses to antibodies that block the binding of pathogenic antibodies to NMDAR (e.g., ART5803).

[0005] In some embodiments, the following are disclosed: a plurality of N-methyl-D-aspartate receptor (NMDAR) antigens coupled to a solid support, wherein the plurality of NMDAR antigens are coupled to the solid support in a non-random manner.

[0006] In some embodiments, the solid support includes plates, test tubes, microtiter wells, beads, slides, membranes, microparticles, nanoparticles, or chips. In some embodiments, the solid support includes plastics, derivatized plastics, polystyrene, polyvinyl chloride, magnetic metals, non-magnetic metals, glass, or silicon. In some embodiments, the multiple NMDAR antigens include the amino acid sequence shown in any of SEQ ID NOs: 1-5. In some embodiments, the multiple NMDAR antigens include the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the multiple NMDAR antigens include the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the multiple NMDAR antigens include the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the multiple NMDAR antigens include the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the multiple NMDAR antigens include the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the multiple NMDAR antigens lack an N-terminal signal peptide. In some embodiments, the multiple NMDAR antigens do not include the GluN2A domain, GluN2B domain, GluN2C domain, GluN2D domain, or any combination thereof of the NMDAR. In some embodiments, multiple NMDAR antigens are covalently coupled to a solid support. In some embodiments, multiple NMDAR antigens are noncovalently coupled to a solid support. In some embodiments, the multiple NMDAR antigens further comprise an Fc polypeptide. In some embodiments, the Fc polypeptide is bound to the C-terminus of the multiple NMDAR antigens. In some embodiments, the Fc polypeptide is bound to the N-terminus of the multiple NMDAR antigens. In some embodiments, the multiple antigens comprise an affinity tag. In some embodiments, the affinity tag comprises a His tag, a FLAG tag, a c-Myc tag, an HA tag, a V5 tag, a GST tag, an MBP tag, a CBP tag, a CBD tag, an Avi- tag, a biotinylated Avi- tag, or a combination thereof. In some embodiments, the affinity tag comprises a His tag. In some embodiments, the affinity tag comprises a biotinylated Avi- tag.In some embodiments, the biotinylated Avi-tag is coupled to a solid support containing avidin. In some embodiments, the avidin is a deglycosylated avidin (e.g., neutraavidin or streptavidin). In some embodiments, the affinity tag includes a mouse Fc tag.

[0007] In some embodiments, affinity tags are coupled to the C-terminus of multiple NMDAR antigens. In some embodiments, affinity tags are coupled to the N-terminus of multiple NMDAR antigens. In some embodiments, mouse Fc tags are non-covalently coupled to a solid support. In some embodiments, His tags are non-covalently coupled to a nickel-containing solid support. In some embodiments, the nickel-containing solid support is a well in an enzyme-linked immunosorbent assay (ELISA) plate. In some embodiments, individual NMDAR antigens of multiple NMDAR antigens non-randomly coupled to a solid support are separated by an average distance of 15 nm or less. In some embodiments, multiple NMDAR antigens are bound to an anti-NMDAR antibody. In some embodiments, multiple NMDAR antigens are bound to a pathogenic anti-NMDAR antibody.

[0008] In this specification, in several embodiments, a method for detecting pathological anti-NMDAR antibodies in a biological sample of an individual is disclosed, the method comprising the steps of contacting the biological sample of an individual with a plurality of NMDAR antigens described in any one of the embodiments described above, and detecting the binding of pathological anti-NMDAR antibodies in the biological sample to the plurality of NMDAR antigens.

[0009] In some embodiments, the biological sample includes blood, plasma, serum, saliva, cell lysate, lymph, amniotic fluid, cerebrospinal fluid, tears, mucus, urine, saliva, amniotic fluid, or sweat. In some embodiments, the biological sample includes blood. In some embodiments, the biological sample includes plasma. In some embodiments, the biological sample includes serum. In some embodiments, the biological sample includes lymph. In some embodiments, the biological sample includes cerebrospinal fluid. In some embodiments, the biological sample includes amniotic fluid. In some embodiments, the binding of pathological anti-NMDAR antibodies to multiple NMDAR antigens is detected by an enzymatic reaction. In some embodiments, the binding of pathological anti-NMDAR antibodies to multiple NMDAR antigens is detected by a fluorescence signal. In some embodiments, the binding of pathological anti-NMDAR antibodies to multiple NMDAR antigens is detected by an immunoassay. In some embodiments, the immunoassay includes an ELISA. In some embodiments, ELISA involves contacting a biological sample of an individual with multiple NMDAR antigens to generate a pathological anti-NMDAR antibody / NMDAR antigen complex, and contacting the pathological anti-NMDAR antibody / NMDAR antigen complex with a detection agent that binds to the pathological anti-NMDAR antibody. In some embodiments, the detection agent is an anti-isotype antibody. In some embodiments, the detection agent is labeled with a fluorescent compound or enzyme. In some embodiments, the binding of the pathological anti-NMDAR antibody to multiple NMDAR antigens is detected by fluorescence-activated cell sorting (FACS). In some embodiments, the anti-NMDAR antibody present in the biological sample is detected at concentrations ranging from 0.3 ng / mL to 100 μg / mL (e.g., 0.3 to 100 ng / mL). In some embodiments, the presence of the pathological anti-NMDAR antibody indicates a diagnosis of autoimmune encephalitis, dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression. In some embodiments, the presence of pathological anti-NMDAR antibodies indicates a diagnosis of autoimmune encephalitis. In some embodiments, the method further includes a step of quantifying the level of pathological anti-NMDAR antibodies in a biological sample. In some embodiments, the method further includes generating a report indicating the presence or level of anti-NMDAR antibodies in a biological sample.In some embodiments, the method further includes administering to an individual a therapeutic anti-NMDAR antibody or an anti-NMDAR-binding fragment thereof, which prevents the binding of pathological anti-NMDAR autoantibodies to NMDAR in the individual, based on the detection of the presence of pathological anti-NMDAR antibodies. In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 13, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 14, and / or a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region (VL) comprising d) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 16, e) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 17, and / or f) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 24, and / or a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 25, and a light chain variable region (VL) comprising d) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 26, e) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence EDN, and / or f) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 28.In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 33, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 34, and / or a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 35, and a light chain variable region (VL) comprising d) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 36, e) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 37, and / or f) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 38. In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 44, and / or a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region (VL) comprising d) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 46, e) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 47, and / or f) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 53, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 54, and / or a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 55, and a light chain variable region (VL) comprising d) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 56, e) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 57, and / or f) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 58.In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 63, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 64, and / or a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 65, and a light chain variable region (VL) comprising d) a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 66, e) a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 67, and / or f) a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 68. In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy-chain immunoglobulin variable region and a light-chain immunoglobulin variable region selected from SEQ ID NOs: 11 and 12, SEQ ID NOs: 21 and 22, SEQ ID NOs: 31 and 32, SEQ ID NOs: 41 and 42, SEQ ID NOs: 51 and 52, or SEQ ID NOs: 61 and 62. In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy-chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NOs: 11 and a light-chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NOs: 12. In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy-chain immunoglobulin variable region containing an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NOs: 11 and a light-chain immunoglobulin variable region containing an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NOs: 12. In some embodiments, the therapeutic anti-NMDAR antibody is a one-arm antibody. In some embodiments, the step of detecting the binding of a pathological anti-NMDAR antibody in a biological sample includes detecting the binding of the IgG of the pathological anti-NMDAR antibody. In some embodiments, the step of detecting the binding of pathological anti-NMDAR antibodies in a biological sample includes detecting the binding of IgA to the pathological anti-NMDAR antibodies. In some embodiments, the step of detecting the binding of pathological anti-NMDAR antibodies in a biological sample includes detecting the binding of IgM to the pathological anti-NMDAR antibodies.In some embodiments, the step of detecting the binding of pathological anti-NMDAR antibodies in a biological sample is independent of whether the pathological anti-NMDAR antibodies in the biological sample are IgA, IgM, or IgG. [Brief explanation of the drawing]

[0010] Novel features described herein are particularly described in the appended claims. The features and their advantages described herein will be better understood by referring to the following detailed description illustrating exemplary examples in which the principles of the features described herein are applied, and by referring to the following appended drawings.

[0011] [Figure 1] Figure 1A shows random coating of NTD-NR1 on a plate, as well as its binding with pathogenic autoantibody 102Ab (two arms) and therapeutic antibody ART5803 (one arm). Figure 1B shows non-randomized coating of NTD-NR1 with a His tag non-covalently coupled to a nickel-containing solid support, as well as its binding with pathogenic autoantibody 102Ab and therapeutic antibody ART5803. [Figure 2A] Figure 2A compares the binding affinity of pathogenic autoantibody 102 Ab and therapeutic antibody ART5803 on an NTD-NR1 randomly coated plate. [Figure 2B] Figure 2B compares the binding affinity of pathogenic autoantibody 102 Ab and therapeutic antibody ART5803 on NTD-NR1 non-randomized coated plates. [Figure 3A] Figure 3A compares the binding affinity of pathogenic autoantibody 102 Ab and selected low-affinity anti-NMDAR encephalitis monoclonal autoantibodies on an NTD-NR1 randomly coated plate. [Figure 3B] Figure 3B compares the binding affinity of pathogenic autoantibody 102 Ab and selected low-affinity anti-NMDAR encephalitis monoclonal autoantibodies on NTD-NR1 non-randomized coated plates. [Figure 4]Figure 4 shows that pathogenic anti-NMDAR autoantibodies sensitive to therapeutic antibody ART5803 treatment may be displaced from NTD-NR1 on an NTD-NR1 non-randomized coated surface by ART5803-Fab. [Figure 5] Figure 5 compares dose-dependent inhibition of ART5803-Fab against pathogenic autoantibody 102Ab and selected low-affinity anti-NMDAR encephalitis monoclonal autoantibodies bound to NTD-NR1 on a non-randomized coated surface. [Figure 6A] Figure 6A shows the detection of various anti-NMDAR encephalitis monoclonal autoantibodies in serum using an exemplary NTD-NR1 non-randomized coated plate described herein. [Figure 6B] Figure 6B shows the detection of various anti-NMDAR encephalitis monoclonal autoantibodies in another biological sample (cerebrospinal fluid (CSF)) using the exemplary NTD-NR1 non-randomized coated plate described herein. [Figure 7A] Figure 7A demonstrates that 102 Ab IgG, 102 Ab IgA, and 102 Ab IgM all induce significant internal translocation of NMDAR(NR1) in cell-based assays, thus indicating pathogenicity. [Figure 7B] Figure 7B demonstrates that ART5803 induces NMDAR(NR1) internal translocation of 102 Ab IgM in a cell-based assay. [Figure 7C] Figure 7C demonstrates that ART5803 induces NMDAR(NR1) internal translocation of 102 Ab IgA in a cell-based assay. [Figure 7D] Figure 7D demonstrates that ART5803 induces internal translocation of 102 Ab IgG to NMDAR(NR1) in a cell-based assay. [Figure 8A] Figure 8A shows the detection of 102Ab IgG, 102Ab IgA, and 102Ab IgM in serum using an exemplary NTD-NR1 non-randomized coated plate described herein. [Figure 8B]Figure 8B shows the detection of 102Ab IgG, 102Ab IgA, and 102Ab IgM in another biological sample (cerebrospinal fluid (CSF)) using an exemplary NTD-NR1 non-randomized coated plate described herein. [Modes for carrying out the invention]

[0012] This specification describes a plurality of N-methyl-D-aspartate receptor (NMDAR) antigens coupled to a solid support, wherein the plurality of NMDAR antigens are coupled to the solid support in a non-random manner. The plurality of N-methyl-D-aspartate receptor (NMDAR) antigens coupled to a solid support can be brought into contact with biological samples of individuals for screening or diagnosis of conditions associated with pathogenic anti-NMDAR antigens. definition

[0013] The following description includes specific details to provide a complete understanding of the various embodiments. However, those skilled in the art will understand that the embodiments provided can be carried out even without these details. Unless otherwise required by context, the term “comprise” and its variations (such as “comprises” and “comprising”) throughout the following specification and claims should be interpreted in an open, comprehensive sense, i.e., “including, but not limited to.” As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural subjects unless the content clearly indicates otherwise. Also note that the term “or” is generally used to include “and / or” unless the content clearly indicates otherwise. Furthermore, the headings provided herein are for convenience only and do not imply any interpretation of the scope or meaning of the embodiments of the claims.

[0014] As used herein, the term “NMDAR,” also known as N-methyl-D-aspartate receptor or NMDA receptor, refers to ion channel-type glutamate receptors and ion channels found in neurons. Ion channel-type glutamate receptors are ligand-gated ion channels that enable rapid ion influx in response to glutamate. Both hyperfunction and hypofunction of NMDARs have been well demonstrated to be associated with neuropsychiatric and neurological disorders.

[0015] As used herein, the terms “NR1” or “NR1 subunit” refer to the NMDAR receptor NR1 subunit. For the purposes of this disclosure, the NR1 subunit comprises the amino acid sequence of SEQ ID NO: 6.

[0016] As used herein, the term "NTD" refers to the N-terminal domain of an NMDAR receptor subunit. An NTD is an extracellular domain involved in assembly and channel regulation. As used herein, an NTD is the NTD of the NR1 subunit. For the purposes of this disclosure, an NTD comprises the amino acid sequence described in any of SEQ ID NOs: 1-5. In certain cases, the NTD lacks an N-terminal signal peptide.

[0017] As used herein, the term “autoantibody” refers to an antibody of biological origin against a component of the same organism, or a binding site having a sequence that binds to an epitope of a protein of the same organism. As used herein, autoantibody refers to a human autoantibody. Pathogenic antibodies as described herein refer to antibodies that bind to NMDARs, induce internal translocation, impair the biological function of NMDARs, and reduce the amount of NMDAR signaling in neurons. Such normal biological function includes activation by glutamate, glycine, or both, and the transport of calcium-containing cations.

[0018] As used herein, “pathological anti-NMDAR antibody” refers to an antibody that binds to NMDAR and induces a disease physiological condition, or in which an individual is predisposed to developing a disease physiological condition. Disease physiological conditions may include anti-NMDAR encephalitis, dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression.

[0019] As used herein, the term “encephalitis” refers to inflammation of the brain accompanied by symptoms including decreased or altered consciousness, personality changes, psychotic delusions, rigidity, headache, fever, confusion, neck rigidity, and vomiting. Complications may include respiratory failure, seizures, hallucinations, dysarthria, memory impairment, and hearing impairment. As used herein, encephalitis is anti-NMDAR encephalitis.

[0020] As used herein, the term "ART5803" refers to an antibody that may be a one-arm antibody having heavy chain and light chain variable regions as shown in Sequence ID No. 11 and 12, respectively. This antibody is described in PCT application WO2021241616A1.

[0021] As used herein, the term “one-arm antibody” differs from a standard antibody in that it is monovalent and contains only a single binding site derived from the heavy chain and light chain variable region. Such a one-arm antibody may still contain constant regions derived from either or both of the heavy chain and / or light chain, and may further pair with a heavy chain that is not further bound to the heavy chain variable region.

[0022] As used herein, the term "about" refers to an approximate amount, within 10% of the stated amount.

[0023] As used herein, the terms “individual,” “patient,” or “subject” refer to an individual diagnosed with at least one disease for which the described compositions and methods are useful for treatment, an individual suspected of having at least one disease, or an individual at risk of developing at least one disease. In certain embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cattle, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0024] The antibodies offered include monoclonal antibodies, multispecific antibodies (e.g., and multireactive antibodies), and antibody fragments. Antibodies include antibody-containing molecules such as antibody conjugates and chimeric molecules. Therefore, antibodies include, but are not limited to, full-length antibodies and native antibodies, as well as fragments and parts thereof that retain their binding specificity, including any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and, but are not limited to, biologically related (antigen-binding) fragments or any specific binding moieties thereof, including those containing Fab, F(ab')2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally one in substantially homogeneous antibody compositions, and therefore any individual antibodies contained within a monoclonal antibody composition are identical except for spontaneous mutations that may be present in small amounts. Monoclonal antibodies may contain the human IgG1 constant region. Monoclonal antibodies may contain the human IgG4 constant region.

[0025] The term “antibody” is used in its broadest sense to include intact antibodies and their functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments, as well as polyclonal and monoclonal antibodies. The term encompasses immunoglobulins such as intrabody, peptide-body, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, as well as genetically engineered and / or otherwise recombinant forms of polyspecific, such as bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term “antibody” should be understood to encompass those functional antibody fragments. The term also encompasses intact or full-length antibodies, including any class or subclass of antibodies, including IgG and its subclasses, IgM, IgE, IgA, and IgD. Antibodies may contain the human IgG1 constant region. Antibodies may contain the human IgG4 constant region.

[0026] Many techniques known in the art can be used to prepare suitable antibodies, such as recombinant antibodies, monoclonal antibodies, or polyclonal antibodies (see, for example, Kohler & Milstein, Nature 256:495-497 (1975), Kozbor et al., Immunology Today 4:72 (1983), Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), Coligan, Current Protocols in Immunology (1991), Harlow & Lane, Antibodies, A Laboratory Manual (1988), and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed. 1986)). Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells, and for example, a gene encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be constructed from hybridomas or plasma cells. Random combinations of heavy and light chain gene products generate a large pool of antibodies with different antigen specificities (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for producing single-chain antibodies or recombinant antibodies (US Patent Nos. 4,946,778 and 4,816,567) may be suitable for producing the antibodies of this disclosure.Furthermore, genetically modified mice or other organisms such as other mammals may be used to humanize or express human antibodies (e.g., U.S. Patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-13 (1994), Fishwild et al., Nature Biotechnology 14:845-51 (1996), Neuberger, Nature Biotechnology 14:826 (1996), and Lonberg & Huszar, See Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, phage display techniques can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen. (See, e.g., McCafferty et al., Nature 348:552-554 (1990), Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can be made bispecific, that is, capable of recognizing two different antigens. (See, e.g., WO93 / 08829, Traunecker et al., EMBO J. 10:3655-3659 (1991), and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently bound antibodies, or immunotoxins. (See, for example, U.S. Patent No. 4,676,980, WO91 / 00360, WO92 / 200373, and EP03089).

[0027] The terms "complementarity determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) has four FRs, and each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4) has four FRs. The precise amino acid sequence boundaries of a given CDR or FR can be found in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J.Mol.Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J.Mol.Biol.262, 732-745.” (“Contact” numbering scheme), and Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan, 27(1):55-77(“IMGT” numbering scheme), Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8, 309(3):657-70, (“Aho” numbering scheme), and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modeling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM” numbering It can be readily determined using one of several well-known schemes, including those described by scheme. In some embodiments, the CDR of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0028] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions enclosed by insertion letters, e.g., "30a," and deletions appearing in some antibodies. The two schemes place specific insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0029] The terms "variable region" or "variable domain" refer to the domain of an antibody heavy or light chain that is involved in binding to an antigen of the antibody. The variable domains of the heavy and light chains of a native antibody (V H and V L ), respectively) generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs (see, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. Further, antibodies that bind to a particular antigen can be isolated using the V L or V H domains from antibodies that bind to the antigen to screen a library of complementary V H or V L domains (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0030] The specific binding or binding of an antibody molecule described herein refers to binding mediated by one or more CDR portions of the antibody. Not all CDRs are required for specific binding. Specific binding can be demonstrated, for example, by showing a significant increase in binding in comparison to an isotype control antibody by ELISA to a particular listed target or antigen.

[0031] As described herein, “epitope” refers to the binding determinant of an antibody or fragment as described herein, which is the minimum necessary for the specific binding of the antibody or fragment to a target antigen. When the target antigen is a polypeptide, the epitope can be continuous or discontinuous. A continuous epitope is formed by one region of the target antigen, while a discontinuous epitope may be formed by two or more distinct regions. A discontinuous epitope may be formed, for example, when the target antigen takes on a tertiary structure in which two amino acid sequences are combined to form a three-dimensional structure to which the antibody binds. When the target antigen is a polypeptide, the epitope is usually a plurality of amino acids linked to the polypeptide chain. A continuous epitope may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids. The epitope may contain a polymer of consecutive amino acids, but not all amino acids in the polymer can come into contact with amino acid residues of the antibody. Such uncontacted amino acids may be important to the structure and linkage of the contacted amino acids and therefore still constitute part of the epitope. Those skilled in the art can determine, for example, whether any given antibody binds to the epitope of a reference antibody by cross-blockage experiments using a reference antibody. In some embodiments, described herein is an antibody that binds to the same epitope as the antibody described. In some embodiments, described herein is an antibody that is competitively blocked by the antibody described. In some embodiments, described herein is an antibody that competes for binding to the antibody described.

[0032] The antibodies provided include antibody fragments. “Antibody fragment” refers to a molecule other than the intact antibody, containing the portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0033] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment containing sequences not found in nature, having two or more antibody regions or chains linked by a synthetic linker, such as a polypeptide linker, and / or not produced by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0034] A “humanized” antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody refers to a variant of a non-human antibody that has been humanized, typically to reduce its immunogenicity against humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0035] The antibodies offered include human antibodies. “Human antibodies” are antibodies that have amino acid sequences corresponding to sequences of antibodies produced by humans or human cells, or non-human sources that utilize a human antibody repertoire including human antibody libraries, or other human antibody coding sequences. This term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those where all or substantially all CDRs are non-human.

[0036] Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus, is extrachromosomal, or is randomly incorporated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Human antibodies can also be derived from human antibody libraries containing antibody coding sequences derived from the human repertoire, including phage displays and cell-free libraries.

[0037] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and are not limited to minimum length. Polypeptides, including antibodies and antibody chains provided, as well as other peptides such as linkers and binding peptides, may contain amino acid residues including native and / or non-native amino acid residues. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may contain modifications to the undenatured or native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as those by site-directed mutagenesis, or accidental, such as those resulting from mutations in the host producing the protein or errors resulting from PCR amplification. In some embodiments, amino acid sequence variants of the antibodies provided herein are intended. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may exist naturally or may be produced synthetically, for example, by modifying one or more of the polypeptide sequences of the present invention and evaluating the biological activity of one or more polypeptides described herein, and / or by using any of several known techniques. For example, amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions of such residues. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has desired characteristics, such as antigen-binding properties.

[0038] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by various known methods in the art, such as using publicly available computer software, including, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0039] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (or, alternatively, a given amino acid sequence A has, or contains, a certain % amino acid sequence identity with (and to) a given amino acid sequence B) is calculated as the fraction X / Y × 100 [wherein X is the number of residues scored by the ALIGN-2 sequence alignment program as matching the alignment identity between A and B in the program, and Y is the total number of residues in B]. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A to B. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0040] In some embodiments, the antibodies provided herein are concentrated in concentrations of approximately 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or less (e.g., 10 nM) relative to the antibody target. -8 Less than M, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M D ) have. In some embodiments, the antibodies provided herein have a strength of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM, or greater (e.g., 10 -8 Less than M, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) dissociation constant (K D) has. The antibody target may be an NR1 target. K D KD can be measured by any suitable assay. In one embodiment, KD can be measured using a surface plasmon resonance assay (e.g., using BIACORE®-2000, BIACORE®-3000, or Octet).

[0041] In some embodiments, the antibodies provided herein may be further modified to include additional known and available non-proteinoid moieties. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / malein anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody can vary, and if two or more polymers are bound, they may be the same molecule or different molecules.

[0042] Antibodies described herein may be encoded by nucleic acids. Nucleic acids are a type of polynucleotide containing two or more nucleotide bases. In some embodiments, nucleic acids are components of a vector that can be used to introduce polynucleotides encoding polypeptides into cells. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a genome-integrated vector or “integration vector” that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an “episome” vector, which is, for example, a nucleic acid capable of extrachromosomal replication. A vector capable of inducing the expression of a manipulably ligated gene is referred herein to as an “expression vector.” Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements such as promoters, enhancers, and polyadenylation signals for use in controlling transcription may be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host (usually conferred by the origin of replication) and select genes to facilitate recognition of transformants may be additionally incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses may be used. Plasmid vectors can be linearized for integration into genomic regions. In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a lentivirus, adenovirus, or adeno-associated virus. In some embodiments, the expression vector is an adenovirus. In some embodiments, the expression vector is an adeno-associated virus. In some embodiments, the expression vector is a lentivirus.

[0043] As used herein, when used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, the terms “homologous,” “homology,” or “percent homology” can be determined using formulas described by Karlin and Altschul (Proc.Natl.Acad.Sci.USA 87:2264-2268, 1990, modified as in Proc.Natl.Acad.Sci.USA 90:5873-5877, 1993). These formulas are incorporated into the basic local alignment search tool (BLAST) program by Altschul et al. (J.Mol.Biol.215:403-410, 1990). Percent homology of sequences can be determined using the latest version of BLAST as of the filing date of this application.

[0044] The nucleic acids encoding antibodies described herein can be used to infect, transfect, transform, or otherwise transgenicize suitable cells, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for antibody production from large cell cultures are known in the art. See Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. 2010 Sep-Oct; 2(5):466-477. In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the mammalian cells are a cell line useful for antibody production, such as Chinese hamster ovary cell (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In some embodiments, the nucleic acid encoding the antibody is incorporated into a genomic locus of a cell useful for antibody production. In some embodiments, a method for producing an antibody is described herein, comprising the step of culturing cells containing the nucleic acid encoding the antibody in vitro under conditions sufficient to enable antibody production and secretion.

[0045] Methods for producing antibodies described herein are also described herein. Such methods include the steps of incubating cells or cell lines containing nucleic acids encoding antibodies in a cell culture medium under conditions sufficient to enable antibody expression and secretion, and further the steps of recovering the antibodies from the cell culture medium. Recovery may further include one or more purification steps to remove live cells, cell debris, non-antibody proteins or polypeptides, undesirable salts, buffers, and culture medium components. In some embodiments, the additional purification steps include centrifugation, ultracentrifugation, purification of protein A, protein G, protein A / G, or protein L, and / or ion exchange chromatography.

[0046] When used herein, “treat,” “treatment,” or “treating” refers, for example, to intentional intervention in a physiological disorder resulting in a reduction of the severity of a disease or illness, a reduction in the duration of the disease course, improvement or elimination of one or more symptoms associated with a disease or illness, or the provision of a beneficial effect to a person with a disease or illness. Treatment does not necessarily have to cure the underlying disease or condition.

[0047] The “therapeutic effective dose,” “effective dose,” “effective amount,” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of the disease, promotes disease regression as demonstrated by a reduction in the severity of disease symptoms, increases the frequency and duration of asymptomatic periods of the disease, or prevents deterioration or impairment due to the disease state. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as by assaying the activity of the drug in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or in in vitro assays.

[0048] As used herein, “pharmaceutically acceptable” with reference to “carrier,” “excipient,” or “diluent” includes any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. In some embodiments, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immune complex, may be coated in a substance to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0049] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. “pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Examples of acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Examples of base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0050] Systems and methods for detecting anti-NMDAR autoantibodies In one embodiment, the disclosure provides a plurality of NMDAR antigens coupled to a support. In some embodiments, the plurality of NMDAR antigens are coupled to the support in a non-random manner.

[0051] In some embodiments, the solid support is a solid support comprising plastic, derivatized plastic, polystyrene, polyvinyl chloride, magnetic metal, non-magnetic metal, glass, nickel, or silicon. In some embodiments, the support is a partially solid support comprising a gel such as agarose gel. In some embodiments, the solid support comprises nickel. In some embodiments, the solid support comprises a plate, test tube, microtiter well, beads, slide, membrane, fine particles, nanoparticles, or chip.

[0052] In some embodiments, the plates include 8-well plates, 12-well plates, 16-well plates, 24-well plates, 48-well plates, 96-well plates, 384-well plates, or 1536-well plates. In some embodiments, the plates include 96-well plates. In some embodiments, the plates include 384-well plates. In some embodiments, the well shapes of the plates include flat-bottomed, round-bottomed, flat-bottomed wells with curved edges at the bottom, or flat-bottomed wells with curved edges at the bottom where eight fins are strategically placed. In some embodiments, the color of the plates is clear, white, or black. The plates may or may not be treated or derivatized to increase the adsorption of polypeptides to their surfaces.

[0053] In some embodiments, the plates include plates coated with antibodies, plates coated with antigens, plates coated with streptavidin, plates coated with NeutrAvidin®, plates coated with nickel, or plates coated with copper chelate. In some embodiments, the plate material includes plastic, derivatized plastic, polystyrene, polyvinyl chloride, glass, or silicon. In some embodiments, the plate surface is hydrophobic. In some embodiments, the plate surface is hydrophilic. Suitable plates for use with the methods of this disclosure include Pierce® maleic anhydride activated plates, Pierce® maleimide activated plates, Nunc® CovaLink® surfaces, Nunc® NucleoLink® strips, or Nunc® Amino® Immobilizer Surfaces.

[0054] In some embodiments, the beads include nickel-coated beads, streptavidin-coated beads, NeutrAvidin®-coated beads, antibody-coated beads, paramagnetic beads, agarose beads, magnetic beads, electrostatic beads, conductive beads, fluorescently labeled beads, colloidal beads, glass beads, semiconductor beads, or polymer beads. In some embodiments, the bead material includes magnetic, agarose, or glass material. In some embodiments, the beads include nickel-coated beads.

[0055] The beads may have an appropriate diameter so that assays, including flow cytometry and immunoprecipitation, can be performed using the beads.

[0056] In some embodiments, the diameter of the beads is approximately 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, and 90 μm. The maximum size is approximately μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

[0057] In some embodiments, the diameter of the beads is at least about 1 μm, at least about 5 μm, at least about 10 μm, at least about 15 μm, at least about 20 μm, at least about 25 μm, at least about 30 μm, at least about 35 μm, at least about 40 μm, at least about 45 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 150 μm, at least about 200 μm, at least about 250 μm, at least about 300 μm, at least about 350 μm, at least about 400 μm, at least about 450 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, or at least about 1000 μm.

[0058] In some embodiments, the diameter of the beads is approximately 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

[0059] In some embodiments, the diameter of the beads is 1-1000 μm, 50-1000 μm, 100-1000 μm, 200-1000 μm, 300-1000 μm, 400-1000 μm, 500-1000 μm, 600-1000 μm, 700-1000 μm, 800-1000 μm, 900-1000 μm, 1-900 μm, 50-900 μm, 100-900 μm, 2 00~900μm, 300~900μm, 400~900μm, 500~900μm, 600~900μm, 700~900μm, 800~900μm, 1~800μm, 50~800 μm, 100~800μm, 200~800μm, 300~800μm, 400~800μm, 500~800μm, 600~800μm, 700~900μm, 1~700μm, 50~ 700μm, 100~700μm, 200~700μm, 300~700μm, 400~700μm, 500~700μm, 600~700μm, 1~600μm, 50~600μm, 100~600μm, 200~600μm, 300~600μm, 400~600μm, 500~600μm, 1~500μm, 50~500μm, 100~500μm, 200~500 The ranges are μm, 300-500 μm, 400-500 μm, 1-400 μm, 50-400 μm, 100-400 μm, 200-400 μm, 300-400 μm, 1-300 μm, 50-300 μm, 100-300 μm, 200-300 μm, 1-200 μm, 50-200 μm, 100-200 μm, 1-100 μm, 50-100 μm, or 1-50 μm.

[0060] In some embodiments, the chips include nickel-coated chips, streptavidin-coated chips, NeutrAvidin®-coated chips, antibody-coated chips, antigen-coated chips, microfluidic chips, or barcoded chips. In some embodiments, the chip material includes ceramic, glass, plastic, derivatized plastic, polystyrene, polyvinyl chloride, gold, chemically inert metals, non-crosslinked carboxylated dextran hydrogels, alkyl derivatives of thiols, disulfides, thioethers, silanes, aminopropyltriethoxysilane, glycidylpropyltrimethoxysilane, or silicon. In some embodiments, the chip shape includes stripes, rings, concentric rings, triangles, rectangles, polyhedra, stars, crossbars, letters, and pictures on planar, convex, concave, or irregular substrates.

[0061] Suitable chips for use with the method of this disclosure include Biacore® Series S Sensor Chip PrismA, Biacore® Series S Sensor Chip NA, Biacore® Chip Protein L, Biacore® Chip Protein G, Biacore® Chip Protein A, Biacore® Chip HPA, Biacore® Chip CM7, Biacore® Chip CM5, Biacore® Chip CM4, Biacore® Chip CM3, Biacore® Chip C1, Biacore® Chip Au, Octet® Streptavidin (SA) Biosensor, Octet® High Precision Streptavidin 2.0 (SAX2) Biosensors, Octet® Anti-Human Fc Capture (AHC) Biosensors, or Octet® Amine Reactive 2nd Generation (AR2G). Biosensors are one example.

[0062] In some embodiments, the antigen is spotted on a chip within an appropriate spot diameter so that assays can be performed using the chip to measure binding affinity, kinetic rate constant, thermodynamics, etc.

[0063] In some embodiments, the spot diameter of the chip is approximately 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, and The thickness is approximately 80 μm, with a maximum of approximately 90 μm, with a maximum of approximately 100 μm, with a maximum of approximately 150 μm, with a maximum of approximately 200 μm, with a maximum of approximately 250 μm, with a maximum of approximately 300 μm, with a maximum of approximately 350 μm, with a maximum of approximately 400 μm, with a maximum of approximately 450 μm, with a maximum of approximately 500 μm, with a maximum of approximately 600 μm, with a maximum of approximately 700 μm, with a maximum of approximately 800 μm, with a maximum of approximately 900 μm, or with a maximum of approximately 1000 μm.

[0064] In some embodiments, the spot diameter of the tip is at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 5 μm, at least about 10 μm, at least about 15 μm, at least about 20 μm, at least about 25 μm, at least about 30 μm, at least about 35 μm, at least about 40 μm, at least about 45 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 150 μm, at least about 200 μm, at least about 250 μm, at least about 300 μm, at least about 350 μm, at least about 400 μm, at least about 450 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, or at least about 1000 μm.

[0065] In some embodiments, the spot diameter of the tip is approximately 0.1 μm, approximately 0.5 μm, approximately 1 μm, approximately 5 μm, approximately 10 μm, approximately 15 μm, approximately 20 μm, approximately 25 μm, approximately 30 μm, approximately 35 μm, approximately 40 μm, approximately 45 μm, approximately 50 μm, approximately 60 μm, approximately 70 μm, approximately 80 μm, approximately 90 μm, approximately 100 μm, approximately 150 μm, approximately 200 μm, approximately 250 μm, approximately 300 μm, approximately 350 μm, approximately 400 μm, approximately 450 μm, approximately 500 μm, approximately 600 μm, approximately 700 μm, approximately 800 μm, approximately 900 μm, or approximately 1000 μm.

[0066] In some embodiments, the spot diameter of the tip is 0.1~1000μm, 1~1000μm, 50~1000μm, 100~1000μm, 200~1000μm, 300~1000μm, 400~1000μm, 500~1000μm, 600~1000μm, 700~1000μm, 800~1000μm, 900~1000μm, 0.1~900μm, 1~900μm, 50~900μm, 100~900μm, 200~900 μm, 300~900μm, 400~900μm, 500~900μm, 600~900μm, 700~900μm, 800~900μm, 0.1~800μm, 1~800μm, 50~800μm, 100~800μm m, 200~800μm, 300~800μm, 400~800μm, 500~800μm, 600~800μm, 700~800μm, 0.1~700μm, 1~700μm, 50~700μm, 100~700μm, 200~700μm, 300~700μm, 400~700μm, 500~700μm, 600~700μm, 0.1~600μm, 1~600μm, 50~600μm, 100~600μm, 200~600μm, 3 00~600μm, 400~600μm, 500~600μm, 0.1~500μm, 1~500μm, 50~500μm, 100~500μm, 200~500μm, 300~500μm, 400~500μm, 0.1 The ranges are ~400μm, 1~400μm, 50~400μm, 100~400μm, 200~400μm, 300~400μm, 0.1~300μm, 1~300μm, 50~300μm, 100~300μm, 200~300μm, 0.1~200μm, 1~200μm, 50~200μm, 100~200μm, 0.1~100μm, 1~100μm, 50~100μm, 0.1~50μm, 1~50μm, or 0.1~1μm.

[0067] In some embodiments, the multiple NMDAR antigens include a single epitope, a single NMDAR epitope, or a single NTD-NR1 epitope. In some embodiments, the NTD-NR1 epitope lacks an N-terminal signal peptide.

[0068] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 1.

[0069] In some embodiments, the NTD-NR1 epitope contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the NTD-NR1 epitope lacks an N-terminal signal peptide.

[0070] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having one or more insertions, deletions, and / or substitutions relative to SEQ ID NO: 1.

[0071] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 40 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 1.

[0072] In some embodiments, the NR1 epitope comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, or up to 40 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 1.

[0073] In some embodiments, the NTD-NR1 epitope includes amino acid insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 relative to SEQ ID NO: 1.

[0074] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0075] In one embodiment, the NTD-NR1 epitope comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 consecutive amino acids derived from SEQ ID NO: 1.

[0076] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 2.

[0077] In some embodiments, the NTD-NR1 epitope comprises the amino acid sequence of SEQ ID NO: 2.

[0078] In some embodiments, the NR1 epitope comprises an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 2.

[0079] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 2.

[0080] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, or up to 30 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 2.

[0081] In some embodiments, the NTD-NR1 epitope includes insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, and 30 relative to SEQ ID NO: 2.

[0082] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0083] In one embodiment, the NTD-NR1 epitope comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 consecutive amino acids derived from SEQ ID NO: 2.

[0084] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 3.

[0085] In some embodiments, the NTD-NR1 epitope comprises the amino acid sequence of SEQ ID NO: 3.

[0086] In some embodiments, the NR1 epitope comprises an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 3.

[0087] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 25 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 3.

[0088] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, or up to 25 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 3.

[0089] In some embodiments, the NTD-NR1 epitope includes insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 relative to SEQ ID NO: 3.

[0090] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0091] In one embodiment, the NTD-NR1 epitope comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 consecutive amino acids derived from SEQ ID NO: 3.

[0092] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 4.

[0093] In some embodiments, the NTD-NR1 epitope comprises the amino acid sequence of SEQ ID NO: 4.

[0094] In some embodiments, the NR1 epitope comprises an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 4.

[0095] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 4.

[0096] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 4.

[0097] In some embodiments, the NTD-NR1 epitope includes insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 relative to SEQ ID NO: 4.

[0098] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0099] In one embodiment, the NTD-NR1 epitope comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids derived from SEQ ID NO: 4.

[0100] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 5.

[0101] In some embodiments, the NTD-NR1 epitope comprises the amino acid sequence of SEQ ID NO: 5.

[0102] In some embodiments, the NR1 epitope comprises an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 5.

[0103] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 5, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 5.

[0104] In some embodiments, the NTD-NR1 epitope comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 5.

[0105] In some embodiments, the NTD-NR1 epitope includes insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 relative to SEQ ID NO: 5.

[0106] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0107] In one embodiment, the NTD-NR1 epitope comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids derived from SEQ ID NO: 5.

[0108] In some embodiments, the NMDAR antigens further comprise an Fc epitope. In some embodiments, the Fc epitope comprises an amino acid sequence having at least about 90%, at least 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence similarity to SEQ ID NO: 19.

[0109] In some embodiments, the Fc epitope comprises the amino acid sequence of SEQ ID NO: 19.

[0110] In some embodiments, the Fc epitope comprises an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 19.

[0111] In some embodiments, the Fc epitope comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 19.

[0112] In some embodiments, the Fc epitope comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 19.

[0113] In some embodiments, the Fc epitope includes insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 relative to sequence number 19.

[0114] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0115] In one embodiment, the Fc peptide contains 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids derived from SEQ ID NO: 19.

[0116] In some embodiments, the Fc peptide is bound to the C-terminus of the NMDAR. In some embodiments, the Fc peptide is bound to the N-terminus of the NMDAR.

[0117] In some embodiments, the multiple NMDAR antigens do not include the GluN2A domain, GluN2B domain, GluN2C domain, GluN2D domain, or any combination thereof of the NMDAR.

[0118] In some embodiments, multiple NMDAR antigens are non-randomly coupled to a solid support by affinity tags. In some embodiments, multiple NMDAR antigens are non-randomly coupled to a solid support by a method other than affinity-based methods.

[0119] In some embodiments, the NMDAR antigens further include affinity tags, which include His tags, FLAG tags, c-Myc tags, HA tags, V5 tags, GST tags, MBP tags, CBP tags, CBD tags, Avi- tags, biotinylated Avi- tags, or combinations thereof.

[0120] In some embodiments, the affinity tag is coupled to the C-terminus of the NMDAR antigen. In some embodiments, the affinity tag is coupled to the N-terminus of the NMDAR antigen.

[0121] In some embodiments, the affinity tag includes a His tag. An exemplary His tag sequence is shown in SEQ ID NO: 70. In some embodiments, the His tag includes multiple histidine residues, e.g., 6, 7, 8, 9, or 10 histidine residues. In some embodiments, the His tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the His tag is bound to the N-terminus of the NMDAR antigen. In some embodiments, the His tag is non-covalently coupled to a nickel-containing solid support. As a non-limiting example, His-tagged antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript.

[0122] In some embodiments, the affinity tag includes a c-Myc tag. An exemplary c-Myc tag sequence is shown in SEQ ID NO: 71. In some embodiments, the c-Myc tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the c-Myc tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, c-Myc-tagged antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abcam, and Cell Signal.

[0123] In some embodiments, the affinity tag includes an HA tag. An exemplary HA tag sequence is shown in SEQ ID NO: 72. In some embodiments, the HA tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the HA tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, HA-tagged antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal, and Abcam.

[0124] In some embodiments, the affinity tag includes a V5 tag. An exemplary V5 tag sequence is shown in SEQ ID NO: 73. In some embodiments, the V5 tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the V5 tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, V5-tagged antibodies are commercially available from suppliers including Abcam, Thermo Fisher, and Sigma-Aldrich.

[0125] In some embodiments, the affinity tag includes a GST tag. An exemplary GST tag sequence is shown in SEQ ID NO: 74. In some embodiments, the GST tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the GST tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, GST-tagged antibodies are commercially available from suppliers including Cube Biotech, Proteintech, and Sigma-Aldrich.

[0126] In some embodiments, the affinity tag includes an MBP tag. An exemplary MBP tag sequence is shown in SEQ ID NO: 75. In some embodiments, the MBP tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the MBP tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, MBP-tagged antibodies are commercially available from suppliers including Thermo Fisher, OriGene, and Sigma-Aldrich.

[0127] In some embodiments, the affinity tag includes a CBP tag. An exemplary CBP tag sequence is shown in SEQ ID NO: 76. In some embodiments, the CBP tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the CBP tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, CBP-tagged antibodies are commercially available from suppliers including Bioss Antibodies, GenScript, and Thermo Fisher.

[0128] In some embodiments, the affinity tag includes a CBD tag. An exemplary CBD tag sequence includes the amino acid sequence shown in SEQ ID NO: 77. In some embodiments, the CBD tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the CBD tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, CBD-tagged antibodies are commercially available from suppliers including New England Biolab and Abcam.

[0129] In some embodiments, the affinity tag includes a biotinylated Avi-tag. An exemplary Avi-tag sequence includes the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the Avi-tag includes a biotinylated Avi-tag. In some embodiments, the Avi-tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the Avi-tag is bound to the N-terminus of the NMDAR antigen. In some embodiments, the biotinylated Avi-tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the biotinylated Avi-tag is bound to the N-terminus of the NMDAR antigen. As a non-limiting example, Avi-tagged antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia.

[0130] In some embodiments, affinity tags include biotinylated Avi-tags and His tags, for example, the Avi-tags and / or His tags disclosed herein.

[0131] In some embodiments, the affinity tag includes an Fc tag. An exemplary Fc tag sequence is shown in SEQ ID NO: 81. In some embodiments, the Fc tag is bound to the C-terminus of the NMDAR antigen. In some embodiments, the Fc tag is bound to the N-terminus of the NMDAR antigen. An exemplary sequence of a mouse Fc tag attached to the C-terminus of NR1-NTD is shown in SEQ ID NO: 82. As a non-limiting example, Fc-tagged antibodies are commercially available from suppliers including Abcam, Thermo Fisher, and Sigma-Aldrich.

[0132] In some embodiments, the individual NMDAR antigens of multiple NMDAR antigens non-randomly coupled to a solid support are spaced apart by an average distance of up to about 25 nm or less, up to about 20 nm, up to about 15 nm, up to about 10 nm, up to about 5 nm, up to about 2 nm, or up to about 1 nm.

[0133] In some embodiments, the individual NMDAR antigens of multiple NMDAR antigens non-randomly coupled to a solid support are separated by an average distance of at least about 25 nm, at least about 20 nm, at least about 15 nm, at least about 10 nm, at least about 5 nm, at least about 2 nm, or at least about 1 nM.

[0134] In some embodiments, the individual NMDAR antigens of multiple NMDAR antigens non-randomly coupled to a solid support are spaced apart by average distances of approximately 25 nm, 20 nm, 15 nm, 10 nm, 5 nm, 2 nm, or 1 nm.

[0135] In some embodiments, the individual NMDAR antigens of multiple NMDAR antigens non-randomly coupled to a solid support are spaced apart by average distances in the range of 1-20 nm, 2-20 nm, 5-20 nm, 10-20 nm, 15-20 nm, 1-18 nm, 2-18 nm, 5-18 nm, 10-18 nm, 15-18 nm, 1-15 nm, 2-15 nm, 5-15 nm, 10-15 nm, 1-10 nm, 2-10 nm, 5-10 nm, 1-5 nm, 2-5 nm, or 1-2 nm.

[0136] In some embodiments, multiple NMDAR antigens are present at a maximum concentration of approximately 1000 ng / cm³. 2 Up to approximately 900 ng / cm² 2 up to approximately 800 ng / cm² 2 Up to approximately 700 ng / cm² 2 up to approximately 600 ng / cm² 2 up to approximately 500 ng / cm² 2 Up to approximately 400 ng / cm² 2 Up to approximately 300 ng / cm² 2 up to approximately 200 ng / cm² 2 up to approximately 100 ng / cm² 2 up to approximately 90 ng / cm² 2 up to approximately 80 ng / cm² 2 up to approximately 70 ng / cm² 2 up to approximately 60 ng / cm² 2 up to approximately 50 ng / cm² 2 up to approximately 40 ng / cm³ 2 up to approximately 30 ng / cm² 2 up to approximately 20 ng / cm³ 2 up to approximately 10 ng / cm² 2 Up to approximately 5 ng / cm² 2 Up to approximately 2 ng / cm³ 2 , or up to approximately 1 ng / cm³ 2 They are coupled non-randomly to the solid support at a density of .

[0137] In some embodiments, multiple NMDAR antigens are present at a concentration of at least about 1000 ng / cm³. 2 , at least about 900 ng / cm 2 at least approximately 800 ng / cm²2 at least approximately 700 ng / cm² 2 at least approximately 600 ng / cm² 2 at least approximately 500 ng / cm² 2 at least approximately 400 ng / cm³ 2 , at least about 300 ng / cm 2 at least approximately 200 ng / cm³ 2 at least about 100 ng / cm³ 2 at least about 90 ng / cm 2 at least approximately 80 ng / cm³ 2 at least about 70 ng / cm 2 at least about 60 ng / cm 2 at least about 50 ng / cm³ 2 at least about 40 ng / cm³ 2 at least about 30 ng / cm 2 at least about 20 ng / cm³ 2 at least about 10 ng / cm³ 2 at least about 5 ng / cm 2 at least about 2 ng / cm³ 2 , or at least about 1 ng / cm 2 They are coupled non-randomly to the solid support at a density of .

[0138] In some embodiments, multiple NMDAR antigens are present at approximately 1000 ng / cm³. 2 , about 900ng / cm 2 , about 800ng / cm 2 , about 700ng / cm 2 , about 600ng / cm 2 , about 500ng / cm 2 , about 400ng / cm 2 , about 300ng / cm 2 , about 200ng / cm 2 , about 100ng / cm 2 , about 90ng / cm 2 , about 80ng / cm 2 , about 70ng / cm 2 , about 60ng / cm 2 , about 50ng / cm 2 , about 40ng / cm 2 , about 30ng / cm2 、at a density of about 20 ng / cm 2 、about 10 ng / cm 2 、about 5 ng / cm 2 、about 2 ng / cm 2 、or about 1 ng / cm 2 and are non - randomly coupled to the solid support.

[0139] In some embodiments, a plurality of NMDAR antigens are at 1 - 1000 ng / cm 2 、2 - 1000 ng / cm 2 、5 - 1000 ng / cm 2 、10 - 1000 ng / cm 2 、20 - 1000 ng / cm 2 、30 - 1000 ng / cm 2 、40 - 1000 ng / cm 2 、50 - 1000 ng / cm 2 、60 - 1000 ng / cm 2 、70 - 1000 ng / cm 2 、80 - 1000 ng / cm 2 、90 - 1000 ng / cm 2 、100 - 1000 ng / cm 2 、200 - 1000 ng / cm 2 、300 - 1000 ng / cm 2 、400 - 1000 ng / cm 2 、500 - 1000 ng / cm 2 、600 - 1000 ng / cm 2 、700 - 1000 ng / cm 2 、800 - 1000 ng / cm 2 、900 - 1000 ng / cm 2 、1 - 900 ng / cm 2 、2 - 900 ng / cm 2 、5 - 900 ng / cm 2 、10 - 900 ng / cm 2 、20 - 900 ng / cm 2 、30 - 900 ng / cm 2 、40 - 900 ng / cm 2 、50 - 900 ng / cm 2 、60 - 900 ng / cm 2 、70 - 900 ng / cm2 、80~900ng / cm 2 、90~900ng / cm 2 、100~900ng / cm 2 、200~900ng / cm 2 、300~900ng / cm 2 、400~900ng / cm 2 、500~900ng / cm 2 、600~900ng / cm 2 、700~900ng / cm 2 、800~900ng / cm 2 、1~800ng / cm 2 、2~800ng / cm 2 、5~800ng / cm 2 、10~800ng / cm 2 、20~800ng / cm 2 、30~800ng / cm 2 、40~800ng / cm 2 、50~800ng / cm 2 、60~800ng / cm 2 、70~800ng / cm 2 、80~800ng / cm 2 、90~800ng / cm 2 、100~800ng / cm 2 、200~800ng / cm 2 、300~800ng / cm 2 、400~800ng / cm 2 、500~800ng / cm 2 、600~800ng / cm 2 、700~800ng / cm 2 、1~700ng / cm 2 、2~700ng / cm 2 、5~700ng / cm 2 、10~700ng / cm 2 、20~700ng / cm 2 、30~700ng / cm 2 、40~700ng / cm 2 、50~700ng / cm 2 、60~700ng / cm 2 、70~700ng / cm 2、80~700ng / cm 2 、90~700ng / cm 2 、100~700ng / cm 2 、200~700ng / cm 2 、300~700ng / cm 2 、400~700ng / cm 2 、500~700ng / cm 2 、600~700ng / cm 2 、1~600ng / cm 2 、2~600ng / cm 2 、5~600ng / cm 2 、10~600ng / cm 2 、20~600ng / cm 2 、30~600ng / cm 2 、40~600ng / cm 2 、50~600ng / cm 2 、60~600ng / cm 2 、70~600ng / cm 2 、80~600ng / cm 2 、90~600ng / cm 2 、100~600ng / cm 2 、200~600ng / cm 2 、300~600ng / cm 2 、400~600ng / cm 2 、500~600ng / cm 2 、1~500ng / cm 2 、2~500ng / cm 2 、5~500ng / cm 2 、10~500ng / cm 2 、20~500ng / cm 2 、30~500ng / cm 2 、40~500ng / cm 2 、50~500ng / cm 2 、60~500ng / cm 2 、70~500ng / cm 2 、80~500ng / cm 2 、90~500ng / cm 2 、100~500ng / cm 2 、200~500ng / cm 2 、300~500ng / cm2 、400~500ng / cm 2 、1~400ng / cm 2 、2~400ng / cm 2 、5~400ng / cm 2 、10~400ng / cm 2 、20~400ng / cm 2 、30~400ng / cm 2 、40~400ng / cm 2 、50~400ng / cm 2 、60~400ng / cm 2 、70~400ng / cm 2 、80~400ng / cm 2 、90~400ng / cm 2 、100~400ng / cm 2 、200~400ng / cm 2 、300~400ng / cm 2 、1~300ng / cm 2 、2~300ng / cm 2 、5~300ng / cm 2 、10~300ng / cm 2 、20~300ng / cm 2 、30~300ng / cm 2 、40~300ng / cm 2 、50~300ng / cm 2 、60~300ng / cm 2 、70~300ng / cm 2 、80~300ng / cm 2 、90~300ng / cm 2 、100~300ng / cm 2 、200~300ng / cm 2 、1~200ng / cm 2 、2~200ng / cm 2 、5~200ng / cm 2 、10~200ng / cm 2 、20~200ng / cm 2 、30~200ng / cm 2 、40~200ng / cm 2 、50~200ng / cm 2 、60~200ng / cm 2 、70~200ng / cm2 、80~200ng / cm 2 、90~200ng / cm 2 、100~200ng / cm 2 、1~100ng / cm 2 、2~100ng / cm 2 、5~100ng / cm 2 、10~100ng / cm 2 、20~100ng / cm 2 、30~100ng / cm 2 、40~100ng / cm 2 、50~100ng / cm 2 、60~100ng / cm 2 、70~100ng / cm 2 、80~100ng / cm 2 、90~100ng / cm 2 、1~90ng / cm 2 、2~90ng / cm 2 、5~90ng / cm 2 、10~90ng / cm 2 、20~90ng / cm 2 、30~90ng / cm 2 、40~90ng / cm 2 、50~90ng / cm 2 、60~90ng / cm 2 、70~90ng / cm 2 、80~90ng / cm 2 、1~80ng / cm 2 、2~80ng / cm 2 、5~80ng / cm 2 、10~80ng / cm 2 、20~80ng / cm 2 、30~80ng / cm 2 、40~80ng / cm 2 、50~80ng / cm 2 、60~80ng / cm 2 、70~80ng / cm 2 、1~70ng / cm 2 、2~70ng / cm 2 、5~70ng / cm 2 、10~70ng / cm 2 、20~70ng / cm 230-70 ng / cm 2 40-70 ng / cm 2 50-70 ng / cm 2 60-70 ng / cm 2 , 1-60 ng / cm 2 , 2-60 ng / cm 2 5-60 ng / cm 2 , 10-60 ng / cm 2 20-60 ng / cm 2 30-60 ng / cm 2 40-60 ng / cm 2 50-60 ng / cm 2 , 1-50 ng / cm 2 , 2-50 ng / cm 2 5-50 ng / cm 2 , 10-50 ng / cm 2 , 20-50 ng / cm 2 30-50 ng / cm 2 40-50 ng / cm 2 , 1-40 ng / cm 2 , 2-40 ng / cm 2 5-40 ng / cm 2 , 10-40 ng / cm 2 , 20-40 ng / cm 2 30-40 ng / cm 2 , 1-30 ng / cm 2 2-30 ng / cm 2 5-30 ng / cm 2 , 10-30 ng / cm 2 20-30 ng / cm 2 , 1-20 ng / cm 2 , 2-20 ng / cm 2 5-20 ng / cm 2 , 10-20 ng / cm 2 , 1-10 ng / cm 2 , 2-10 ng / cm 2 5-10 ng / cm 2 , 1-5 ng / cm 2 , 2-5 ng / cm 2 , or 1-2 ng / cm 2 It is coupled non-randomly to the solid support at a density within the range of [value].

[0140] In one embodiment, the present disclosure provides a method for detecting pathological anti-NMDAR antibodies in a biological sample of an individual, comprising the steps of: contacting the biological sample of the individual with a plurality of NMDAR antigens described herein; and detecting the binding of the pathological anti-NMDAR antibodies in the biological sample to the plurality of NMDAR antigens.

[0141] In some embodiments, the biological sample includes blood, plasma, serum, saliva, cell lysate, lymph, amniotic fluid, cerebrospinal fluid, tears, mucus, urine, saliva, amniotic fluid, or sweat. In some embodiments, the biological sample includes plasma, serum, or cerebrospinal fluid. In some embodiments, the biological sample includes blood, plasma, serum, lymph, or cerebrospinal fluid. In some embodiments, the biological sample includes amniotic fluid. In some embodiments, the biological sample includes blood. In some embodiments, the biological sample includes plasma. In some embodiments, the biological sample includes serum. In some embodiments, the biological sample includes lymph. In some embodiments, the biological sample includes cerebrospinal fluid. In some embodiments, the biological sample further includes a diluent. In some embodiments, the biological sample includes any suitable amount of body fluid or tissue (e.g., blood, saliva, urine, skin, or other tissue) that can be collected from an individual. In some embodiments, the biological sample includes a portion of the undiluted sample in a suitable diluent, for example, up to 50%, 40%, 30%, 20%, 10%, 5%, or 1% by weight or volume of the sample included in the assay for the anti-NMDAR antibody.

[0142] In some embodiments, the binding of pathological anti-NMDAR antibodies in a biological sample to multiple NMDAR antigens is detected by an enzymatic reaction. In some embodiments, the enzymatic reaction includes enzymes, which include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (Gal), glucose oxidase, luciferase, β-lactamase, urease, or lysozyme. In some embodiments, the enzyme includes HRP. In some embodiments, the enzyme includes HRP and AP. In some embodiments, the enzymatic reaction includes a substrate, which includes TMB (3,3',5,5'-tetramethylbenzidine), ABTS (2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), OPD (o-phenylenediamine dihydrochloride), PNPP (p-nitrophenyl phosphate), ONPG (o-nitrophenyl-β-D-galactopyranoside), or AMPPD (adamantyl 1,2-dioxetanephenyl phosphate).

[0143] In some embodiments, the binding of pathological anti-NMDAR antibodies to multiple NMDAR antigens in a biological sample is detected by a fluorescence signal. In some embodiments, the fluorescence signal is generated by fluorophores, which include Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, Coumarin, Cy3, Cy5, Fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, PE-Cyanine7, PerCP-Cyanine5.5, Tetramethylrhodamine (TRITC), Texas Red, eFluor 450, eFluor 506, eFluor 660, PE-eFluor 610, and PerCP-eFluor 710, APC-eFluor 780, Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, Super Bright 780, Brilliant Ultra Violet Polymer Dyes, Brilliant Ultra Violet 395, Brilliant Ultra Violet 496, Brilliant Ultra Violet 563, Brilliant Ultra Violet 615, Brilliant Ultra Violet 661, Brilliant Ultra Violet 737, Brilliant Ultra Violet 805, Brilliant Violet 421, Brilliant Violet 480, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet786, NovaFluor Blue 510 Dye, NovaFluor Blue 530 Dye, NovaFluor Blue 555 Dye, NovaFluor Blue 585 Dye, NovaFluor Blue 610-30S Dye, NovaFluor Blue 610-70S Dye, NovaFluor Blue 660-40S Dye, NovaFluor Blue 690, NovaFluor Blue 660-120S Dye, NovaFluor Yellow 570 Dye, NovaFluor Yellow 590 Dye, NovaFluor Yellow 610 Dye, NovaFluor Yellow 660 Dye, NovaFluor Yellow 690 Dye, NovaFluor Yellow 700 Dye, NovaFluor Yellow 730 Dye, NovaFluor Yellow 755, NovaFluor Red 660 Dye, NovaFluor Red 685 Dye, NovaFluor Red 700 Dye, NovaFluor Red 710 Includes Dye, NovaFluor Red 725, or NovaFluor Red 755.

[0144] In some embodiments, the binding of pathological anti-NMDAR antibodies in a biological sample to multiple NMDAR antigens is detected by immunoassay. In some embodiments, the immunoassay includes enzyme immunoassay, radioimmunoassay, fluoroimmunoassay, chemiluminescence immunoassay, counting immunoassay, immunoenzyme assay, enzyme multiplication immunoassay technology, photoacoustic immunoassay, real-time immunoquantification PCR, or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the immunoassay includes ELISA.

[0145] In some embodiments, ELISA includes contacting a biological sample of an individual with multiple NMDAR antigens to produce a pathological anti-NMDAR antibody / NMDAR antigen complex, and contacting the pathological anti-NMDAR antibody / NMDAR antigen complex with a detection agent that binds to the pathological anti-NMDAR antibody.

[0146] In some embodiments, the detection agent includes an antibody. In some embodiments, the antibody targets ART5803. In some embodiments, the antibody agent includes an anti-isotype antibody. In some embodiments, the anti-isotype antibody includes IgA, IgD, IgG, IgE, or IgM that targets the antibody. In some embodiments, the anti-isotype antibody includes IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, IgE, or IgM that targets the antibody. In some embodiments, the anti-isotype antibody includes IgA, IgG, or IgM that targets the antibody. In some embodiments, the anti-isotype antibody includes IgG that targets the antibody. In some embodiments, the anti-isotype antibody includes IgG1 that targets the antibody. In some embodiments, the anti-isotype antibody includes IgG2 that targets the antibody. In some embodiments, the anti-isotype antibody includes IgG3 that targets the antibody. In some embodiments, the anti-isotype antibody includes IgG4 that targets the antibody. In some embodiments, the anti-isotype antibody includes IgA that targets the antibody. In some embodiments, the anti-isotype antibody includes IgA1 targeting the antibody. In some embodiments, the anti-isotype antibody includes IgA2 targeting the antibody. In some embodiments, the anti-isotype antibody includes IgM targeting the antibody.

[0147] In some embodiments, the detection agent is labeled with a fluorescent tag, a radioactive tag, or an enzyme.

[0148] In some embodiments, the fluorescent tag comprises a fluorescent protein, which includes yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, cyanine, dansyl chloride, phycocyanin, Venus, citrin, mRuby3, mCherry, tdTomato, dsRed, phycoerythrin, or a combination thereof. In some embodiments, the fluorescent tag comprises a fluorophore disclosed herein.

[0149] In some embodiments, the radioactive tag is 32 P, 33 P, 47 Sc, 59 Fe, 64 Cu, 67 Cu, 75Se, 77 As, 89 Sr, 90 Y, 99 Mo, 105 Rh, 109 Pd, 111 Ag, 125 I, 131 I, 142 Pr, 143 Pr, 149 PM, 153 Sm, 161 Tb, 166 Ho, 169 Er. 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 Pb, 213 Bi, 58 Co, 67 Ga, 80m Br, 99m Tc, 193m Rh, 109 Pt, 111 In, 119 Sb, 125 I, 161 Ho,189m Os, 192 Ir, 152 Dy, 211 At, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 225 Ac, 221 Fr, 217 At, 213 Bi, 225 This includes Fm, or any combination thereof.

[0150] In some embodiments, the enzyme comprises horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (Gal), glucose oxidase, luciferase, β-lactamase, urease, or lysozyme. In some embodiments, the enzyme comprises HRP. In some embodiments, the enzyme comprises HRP and AP.

[0151] In some embodiments, the binding of pathological anti-NMDAR antibodies to multiple NMDAR antigens is detected by fluorescence-activated cell sorting (FACS). In some embodiments, FACS includes contacting the biological sample of an individual with multiple NMDAR antigens expressed in HEK cells to produce pathological anti-NMDAR antibody / NMDAR antigen complexes, and contacting the pathological anti-NMDAR antibody / NMDAR antigen complexes with a detection agent that binds to the pathological anti-NMDAR antibodies. In some embodiments, the detection agent is disclosed herein.

[0152] In some embodiments, pathological anti-NMDAR antibodies present in the biological sample are detected at concentrations of up to approximately 500 μg / ml, up to approximately 400 μg / ml, up to approximately 300 μg / ml, up to approximately 200 μg / ml, up to approximately 100 μg / ml, up to approximately 90 μg / ml, up to approximately 80 μg / ml, up to approximately 70 μg / ml, up to approximately 60 μg / ml, up to approximately 50 μg / ml, up to approximately 40 μg / ml, up to approximately 30 μg / ml, up to approximately 20 μg / ml, up to approximately 10 μg / ml, up to approximately 5 μg / ml, up to approximately 2 μg / ml, or up to approximately 1 μg / ml, or at higher concentrations. In some embodiments, pathological anti-NMDAR antibodies present in the biological sample are detected at concentrations of up to approximately 500 ng / ml, up to approximately 400 ng / ml, up to approximately 300 ng / ml, up to approximately 200 ng / ml, up to approximately 100 ng / ml, up to approximately 90 ng / ml, up to approximately 80 ng / ml, up to approximately 70 ng / ml, up to approximately 60 ng / ml, up to approximately 50 ng / ml, up to approximately 40 ng / ml, up to approximately 30 ng / ml, up to approximately 20 ng / ml, up to approximately 10 ng / ml, up to approximately 5 ng / ml, up to approximately 2 ng / ml, or up to approximately 1 ng / ml, or at higher concentrations.

[0153] In some embodiments, pathological anti-NMDAR antibodies present in the biological sample are detected at concentrations of at least approximately 500 ng / ml, at least approximately 400 ng / ml, at least approximately 300 ng / ml, at least approximately 200 ng / ml, at least approximately 100 ng / ml, at least approximately 90 ng / ml, at least approximately 80 ng / ml, at least approximately 70 ng / ml, at least approximately 60 ng / ml, at least approximately 50 ng / ml, at least approximately 40 ng / ml, at least approximately 30 ng / ml, at least approximately 20 ng / ml, at least approximately 10 ng / ml, at least approximately 5 ng / ml, at least approximately 2 ng / ml, or at least approximately 1 ng / ml.

[0154] In some embodiments, pathological anti-NMDAR antibodies present in biological samples are detected at concentrations of approximately 500 ng / ml or less, approximately 400 ng / ml, approximately 300 ng / ml, approximately 200 ng / ml, approximately 100 ng / ml, approximately 90 ng / ml, approximately 80 ng / ml, approximately 70 ng / ml, approximately 60 ng / ml, approximately 50 ng / ml, approximately 40 ng / ml, approximately 30 ng / ml, approximately 20 ng / ml, approximately 10 ng / ml, approximately 5 ng / ml, approximately 2 ng / ml, or approximately 1 ng / ml. In some embodiments, pathological anti-NMDAR antibodies present in biological samples are detected at concentrations of approximately 500 μg / ml or less, approximately 400 μg / ml, approximately 300 μg / ml, approximately 200 μg / ml, approximately 100 μg / ml, approximately 90 μg / ml, approximately 80 μg / ml, approximately 70 μg / ml, approximately 60 μg / ml, approximately 50 μg / ml, approximately 40 μg / ml, approximately 30 μg / ml, approximately 20 μg / ml, approximately 10 μg / ml, approximately 5 μg / ml, approximately 2 μg / ml, or approximately 1 μg / ml.

[0155] In some embodiments, pathological anti-NMDAR antibodies present in biological samples are detected at concentrations ranging from 1 ng / ml to 500 μg / ml, 2 ng / ml to 500 μg / ml, 5 ng / ml to 500 μg / ml, 10 ng / ml to 500 μg / ml, 20 ng / ml to 500 μg / ml, 30 ng / ml to 500 μg / ml, 40 ng / ml to 500 μg / ml, 50 ng / ml to 500 μg / ml, 60 ng / ml to 500 μg / ml, 70 ng / ml to 500 μg / ml, 80 ng / ml to 500 μg / ml, 90 ng / ml to 500 μg / ml, 100 ng / ml to 500 μg / ml, or 200 ng / ml to 500 μg / ml. In some embodiments, the pathological anti-NMDAR antibodies present in the biological sample are 1-500 ng / ml, 2-500 ng / ml, 5-500 ng / ml, 10-500 ng / ml, 20-500 ng / ml, 30-500 ng / ml, 40-500 ng / ml, 50-500 ng / ml, 60-500 ng / ml, 70-500 ng / ml, 80-500 ng / ml, and 90-500 ng / ml. l, 100~500ng / ml, 200~500ng / ml, 300~500ng / ml, 400~500ng / ml, 1~400ng / ml, 2~400ng / ml, 5~400ng / ml, 1 0~400ng / ml, 20~400ng / ml, 30~400ng / ml, 40~400ng / ml, 50~400ng / ml, 60~400ng / ml, 70~400ng / ml, 80~400 ng / ml, 90~400ng / ml, 100~400ng / ml, 200~400ng / ml, 300~400ng / ml, 1~300ng / ml, 2~300ng / ml, 5~300ng / m l, 10~300ng / ml, 20~300ng / ml, 30~300ng / ml, 40~300ng / ml, 50~300ng / ml, 60~300ng / ml, 70~300ng / ml, 80 ~300ng / ml, 90~300ng / ml, 100~300ng / ml, 200~300ng / ml, 1~200ng / ml, 2~200ng / ml, 5~200ng / ml, 10~200n g / ml, 20~200ng / ml, 30~200ng / ml, 40~200ng / ml, 50~200ng / ml, 60~200ng / ml, 70~200ng / ml, 80~200ng / ml,Detected at concentrations in the range of 90 - 200 ng / ml, 100 - 200 ng / ml, 1 - 100 ng / ml, 2 - 100 ng / ml, 5 - 100 ng / ml, 10 - 100 ng / ml, 20 - 100 ng / ml, 30 - 100 ng / ml, 40 - 100 ng / ml, 50 - 100 ng / ml, 60 - 100 ng / ml, 70 - 100 ng / ml, 80 - 100 ng / ml, 90 - 100 ng / ml, 1 - 90 ng / ml, 2 - 90 ng / ml, 5 - 90 ng / ml, 10 - 90 ng / ml, 20 - 90 ng / ml, 30 - 90 ng / ml, 40 - 90 ng / ml, 50 - 90 ng / ml, 60 - 90 ng / ml, 70 - 90 ng / ml, 80 - 90 ng / ml, 1 - 80 ng / ml, 2 - 80 ng / ml, 5 - 80 ng / ml, 10 - 80 ng / ml, 20 - 80 ng / ml, 30 - 80 ng / ml, 40 - 80 ng / ml, 50 - 80 ng / ml, 60 - 80 ng / ml, 70 - 80 ng / ml, 1 - 70 ng / ml, 2 - 70 ng / ml, 5 - 70 ng / ml, 10 - 70 ng / ml, 20 - 70 ng / ml, 30 - 70 ng / ml, 40 - 70 ng / ml, 50 - 70 ng / ml, 60 - 70 ng / ml, 1 - 60 ng / ml, 2 - 60 ng / ml, 5 - 60 ng / ml, 10 - 60 ng / ml, 20 - 60 ng / ml, 30 - 60 ng / ml, 40 - 60 ng / ml, 50 - 60 ng / ml, 1 - 50 ng / ml, 2 - 50 ng / ml, 5 - 50 ng / ml, 10 - 50 ng / ml, 20 - 50 ng / ml, 30 - 50 ng / ml, 40 - 50 ng / ml, 1 - 40 ng / ml, 2 - 40 ng / ml, 5 - 40 ng / ml, 10 - 40 ng / ml, 20 - 40 ng / ml, 30 - 40 ng / ml, 1 - 30 ng / ml, 2 - 30 ng / ml, 5 - 30 ng / ml, 10 - 30 ng / ml, 20 - 30 ng / ml, 1 - 20 ng / ml, 2 - 20 ng / ml, 5 - 20 ng / ml, 10 - 20 ng / ml, 1 - 10 ng / ml, 2 - 10 ng / ml, 5 - 10 ng / ml, 1 - 5 ng / ml, 2 - 5 ng / ml, or 1 - 2 ng / ml.,

[0156] In some embodiments, the detection of pathological anti-NMDAR antibodies suggests a diagnosis of anti-NMDAR pathology. In some embodiments, anti-NMDAR pathology includes autoimmune encephalitis, anti-NMDAR autoimmune encephalitis, dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression. In some embodiments, anti-NMDAR pathology includes anti-NMDAR autoimmune encephalitis. In some embodiments, anti-NMDAR pathology includes dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression. In some embodiments, anti-NMDAR pathology includes dementia. In some embodiments, anti-NMDAR pathology includes psychosis. In some embodiments, anti-NMDAR pathology includes schizophrenia. In some embodiments, anti-NMDAR pathology includes bipolar disorder. In some embodiments, anti-NMDAR pathology includes seizures. In some embodiments, anti-NMDAR pathology includes epilepsy. In some embodiments, anti-NMDAR pathology includes depression.

[0157] In some embodiments, following the detection of pathological anti-NMDAR antibodies, there is administration of a therapeutic agent that treats anti-NMDAR pathology. In some embodiments, following the detection of pathological anti-NMDAR antibodies, there is administration of a therapeutic antibody that treats anti-NMDAR condition. In some embodiments, following the detection of pathological anti-NMDAR antibodies, there is administration of a therapeutic anti-NMDAR antibody.

[0158] Therapeutic methods for diseases associated with anti-NMDAR autoantibodies The methods for detecting anti-NMDAR autoantibodies described herein can be combined with steps performed to treat anti-NMDAR pathology.

[0159] In one aspect, based on the detection of the presence of pathological anti-NMDAR antibodies, a therapeutic anti-NMDAR antibody or an anti-NMDAR binding fragment thereof that prevents the binding of pathological anti-NMDAR autoantibodies to an individual's NMDA receptor is disclosed herein.

[0160] In some embodiments, anti-NMDAR pathologies include autoimmune encephalitis, anti-NMDAR autoimmune encephalitis, dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression. In some embodiments, anti-NMDAR pathologies include anti-NMDAR autoimmune encephalitis. In some embodiments, anti-NMDAR pathologies include dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression. In some embodiments, anti-NMDAR pathologies include dementia. In some embodiments, anti-NMDAR pathologies include psychosis. In some embodiments, anti-NMDAR pathologies include schizophrenia. In some embodiments, anti-NMDAR pathologies include bipolar disorder. In some embodiments, anti-NMDAR pathologies include seizures. In some embodiments, anti-NMDAR pathologies include epilepsy. In some embodiments, anti-NMDAR pathologies include depression.

[0161] Treatment refers to a method that attempts to partially or completely relieve, improve, ease, delay the onset, inhibit or slow the progression, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of the particular disease, disorder, and / or illness being treated. Treatment of anti-NMDAR autoimmune encephalitis with a therapeutic anti-NMDAR antibody or its anti-NMDAR binding fragment is expected to result in significant improvement in one or more of the typical clinical symptoms such as improvement of acute mental symptoms, improvement of memory function, reduction of seizures or dyskinesia, and improvement of consciousness and awareness. Various means and tools for assessing mental state are well known to clinicians trained in the art. In some embodiments, clinical improvement before and / or after treatment is determined by the treating physician, generally using the Clinical Global Impression (CGI) as a summary measure of severity (CGI-S) and improvement (CGI-I). In some embodiments, after treatment, the CGI reflects improvement in the mental state. In some embodiments, after treatment, the CGI is significantly improved. In some embodiments, after treatment, the CGI is very significantly improved.

[0162] In some embodiments, clinical improvement in functional recovery before and / or after treatment is assessed using a modified Rankin Scale (mRS), and the mRS score improves after treatment. In some embodiments, the mRS is 4 or less after treatment. In some embodiments, the mRS is 3 or less after treatment. In some embodiments, the mRS is 2 or less after treatment. In some embodiments, the mRS is 1 or less after treatment. In some embodiments, the mRS is 0 after treatment. In some embodiments, the individual receiving treatment has a score 1, 2, 3, 4, 5, or 6 points lower compared to the pre-treatment assessment.

[0163] Clinical improvement in cognitive assessments before and / or after treatment is demonstrated by monitoring the Montreal Cognitive Assessment Score (MOCA), with the treatment resulting in an increase in the MOCA score. In some embodiments, the post-treatment MOCA reflects normal cognitive function. In some embodiments, the post-treatment MOCA is 27 or higher. In some embodiments, the post-treatment MOCA is 26 or higher. In some embodiments, the post-treatment MOCA is 25 or higher. In some embodiments, the post-treatment MOCA is 24 or higher. In some embodiments, the post-treatment MOCA is 23 or higher. In some embodiments, the post-treatment MOCA is 22 or higher. In some embodiments, the post-treatment MOCA is 21 or higher. In some embodiments, the post-treatment MOCA is 20 or higher. In some embodiments, the post-treatment MOCA is 19 or higher. In some embodiments, the post-treatment MOCA is 18 or higher. In some embodiments, the individual receiving treatment has a score 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 points higher compared to the pre-treatment assessment.

[0164] In some embodiments, clinical improvement in patients with seizures before and / or after treatment is demonstrated by a reduction in the number of seizures after treatment. In some embodiments, the number of seizures decreases by 90% or more after treatment. In some embodiments, the number of seizures decreases by 80% or more after treatment. In some embodiments, the number of seizures decreases by 70% or more after treatment. In some embodiments, the number of seizures decreases by 60% or more after treatment. In some embodiments, the number of seizures decreases by 50% or more after treatment. In some embodiments, the number of seizures decreases by 40% or more after treatment. In some embodiments, the number of seizures decreases by 30% or more after treatment. In some embodiments, the number of seizures decreases by 20% or more after treatment. In some embodiments, the number of seizures decreases by 10% or more after treatment. In some embodiments, the number of seizures decreases by 5% or more after treatment.

[0165] In some embodiments, clinical improvement in dyskinesia symptoms before and / or after treatment is assessed using the Abnormal Involuntary Movement Scale (AIMS), Integrated Parkinson's Disease Rating Scale (UPDRS) Part IV, Obeso Dyskinesia Rating Scale, Rush Dyskinesia Rating Scale, Clinical Dyskinesia Rating Scale (CDRS), Lang-Fern Activities of Daily Living Dyskinesia Scale, Parkinson's Disease Dyskinesia Scale (PDYS-26), and Integrated Dyskinesia Rating Scale (UDysRS). In some embodiments, dyskinesia symptoms improve from severe to moderate after treatment. In some embodiments, dyskinesia symptoms improve from severe to mild after treatment. In some embodiments, dyskinesia symptoms improve from severe to minimal after treatment. In some embodiments, dyskinesia symptoms improve from severe to asymptomatic after treatment. In some embodiments, dyskinesia symptoms improve from moderate to mild after treatment. In some embodiments, dyskinesia symptoms improve to moderate to minimal after treatment. In some embodiments, dyskinesia symptoms improve to moderate to none after treatment. In some embodiments, dyskinesia symptoms improve to mild to minimal after treatment. In some embodiments, dyskinesia symptoms improve to mild to asymptomatic after treatment. In some embodiments, dyskinesia symptoms improve to minimal to asymptomatic after treatment.

[0166] In some embodiments, clinical improvement in memory loss before and / or after treatment is assessed using tests such as a mini-cognitive test and a post-treatment mini-mental state test (MMSE). In some embodiments, the MMSE reflects normal cognitive function after treatment. In some embodiments, the MOCA is 29 or higher after treatment. In some embodiments, the MOCA is 28 or higher after treatment. In some embodiments, the MOCA is 27 or higher after treatment. In some embodiments, the MOCA is 26 or higher after treatment. In some embodiments, the MOCA is 25 or higher after treatment. In some embodiments, the MOCA is 24 or higher after treatment. In some embodiments, the MOCA is 23 or higher after treatment. In some embodiments, the MOCA is 22 or higher after treatment. In some embodiments, the MOCA is 21 or higher after treatment. In some embodiments, the MOCA is 20 or higher after treatment. In some embodiments, the MOCA is 19 or higher after treatment. In some embodiments, the individuals receiving the treatment score 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 points or more compared to their pre-treatment evaluation.

[0167] In some embodiments, clinical improvement in schizophrenia before and / or after treatment is assessed using the total score of the Positive and Negative Symptoms Scale (PANSS), the scores of the PANSS positive symptom subscales, the scores of the PANSS negative symptom subscales, the PANSS global psychopathology subscale, the total score of the Brief Psychiatric Rating Scale (BPRS) derived from the PANSS, the BPRS psychosis derived from the PANSS, or any combination thereof. In some embodiments, the PANSS total score decreases by 90% or more after treatment. In some embodiments, the PANSS total score decreases by 80% or more after treatment. In some embodiments, the PANSS total score decreases by 70% or more after treatment. In some embodiments, the PANSS total score decreases by 60% or more after treatment. In some embodiments, the PANSS total score decreases by 50% or more after treatment. In some embodiments, the PANSS total score decreases by 40% or more after treatment. In some embodiments, the PANSS total score decreases by 30% or more after treatment. In some embodiments, the PANSS total score decreases by 20% or more after treatment. In some embodiments, the PANSS total score decreases by 10% or more after treatment. In some embodiments, the PANSS total score decreases by 5% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 90% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 80% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 70% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 60% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 50% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 40% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 30% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 20% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 10% or more after treatment. In some embodiments, the PANSS positive subscale score decreases by 5% or more after treatment.In some embodiments, the PANSS negative subscale score decreases by 90% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 80% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 70% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 60% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 50% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 40% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 30% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 20% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 10% or more after treatment. In some embodiments, the PANSS negative subscale score decreases by 5% or more after treatment. In some embodiments, the BPRS total score decreases by 90% or more after treatment. In some embodiments, the BPRS total score decreases by 80% or more after treatment. In some embodiments, the total BPRS score decreases by 70% or more after treatment. In some embodiments, the total BPRS score decreases by 60% or more after treatment. In some embodiments, the total BPRS score decreases by 50% or more after treatment. In some embodiments, the total BPRS score decreases by 40% or more after treatment. In some embodiments, the total BPRS score decreases by 30% or more after treatment. In some embodiments, the total BPRS score decreases by 20% or more after treatment. In some embodiments, the total BPRS score decreases by 10% or more after treatment. In some embodiments, the total BPRS score decreases by 5% or more after treatment.

[0168] In some embodiments, clinical improvement in depression before and / or after treatment is assessed using the Montgomery-Asberg Depression Rating Scale (MADRS). In some embodiments, the MADRS total score decreases by 90% or more after treatment. In some embodiments, the MADRS total score decreases by 80% or more after treatment. In some embodiments, the MADRS total score decreases by 70% or more after treatment. In some embodiments, the MADRS total score decreases by 60% or more after treatment. In some embodiments, the MADRS total score decreases by 50% or more after treatment. In some embodiments, the MADRS total score decreases by 40% or more after treatment. In some embodiments, the MADRS total score decreases by 30% or more after treatment. In some embodiments, the MADRS total score decreases by 20% or more after treatment. In some embodiments, the MADRS total score decreases by 10% or more after treatment. In some embodiments, the MADRS total score decreases by 5% or more after treatment.

[0169] With respect to anti-NMDAR encephalitis, treatment includes, but is not limited to, administering to an individual a therapeutic anti-NMDAR antibody or an anti-NMDAR conjugate fragment that prevents the binding of pathological anti-NMDAR autoantibodies to the individual's NMDA receptor, based on the detection of the presence of pathological anti-NMDAR antibodies. In some embodiments, the therapeutic anti-NMDAR antibody comprises a one-arm anti-human NR1 antibody. In some embodiments, the therapeutic anti-NMDAR antibody reduces the internal translocation of NMDAR in the presence of a pathogenic anti-NMDAR antibody, where the pathogenic anti-NMDAR antibody comprises 102 Ab. In some embodiments, the therapeutic anti-NMDAR antibody reduces the internal migration of NMDAR by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the presence of a pathogenic anti-NMDAR antibody, the pathogenic anti-NMDAR antibody comprising 102 Ab. In some embodiments, the one-arm anti-human NR1 antibody comprises ART5803.

[0170] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 13, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 14, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region (VL) comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 16, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 17, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 18.

[0171] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 11.

[0172] In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 11.

[0173] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 11.

[0174] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region which includes a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 11.

[0175] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, or up to 45 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 11.

[0176] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 45 relative to SEQ ID NO: 11.

[0177] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0178] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 12.

[0179] In some embodiments, the therapeutic anti-NMDAR antibody includes a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 12.

[0180] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 12.

[0181] In some embodiments, a therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 22 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 12.

[0182] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region having an amino acid sequence with up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, or up to 22 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 12.

[0183] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region having an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 22 insertions, deletions, and / or substitutions relative to SEQ ID NO: 12.

[0184] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. One or more insertions can be continuous, discontinuous, or a combination thereof.

[0185] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 25, and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 26, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence EDN, and a light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 28.

[0186] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 21.

[0187] In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy-chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 21.

[0188] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 21.

[0189] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 insertions, deletions, and / or substitutions relative to SEQ ID NO: 21.

[0190] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, or up to 13 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 21.

[0191] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 relative to SEQ ID NO: 21.

[0192] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0193] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 22.

[0194] In some embodiments, the therapeutic anti-NMDAR antibody includes a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 22.

[0195] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 22.

[0196] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 insertions, deletions, and / or substitutions relative to SEQ ID NO: 22.

[0197] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 22.

[0198] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 relative to SEQ ID NO: 22.

[0199] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0200] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 33, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 34, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 35, and a light chain variable region (VL) comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 36, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 37, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 38.

[0201] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 31.

[0202] In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy-chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 31.

[0203] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 31.

[0204] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 insertions, deletions, and / or substitutions relative to SEQ ID NO: 31.

[0205] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, or up to 13 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 31.

[0206] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 relative to SEQ ID NO: 31.

[0207] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0208] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 32.

[0209] In some embodiments, the therapeutic anti-NMDAR antibody includes a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 32.

[0210] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 32.

[0211] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 insertions, deletions, and / or substitutions relative to SEQ ID NO: 32.

[0212] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 32.

[0213] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 relative to SEQ ID NO: 32.

[0214] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0215] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 44, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region (VL) comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 46, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 48.

[0216] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 41.

[0217] In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy-chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 41.

[0218] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 41.

[0219] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 insertions, deletions, and / or substitutions relative to SEQ ID NO: 41.

[0220] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, or up to 13 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 41.

[0221] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 relative to SEQ ID NO: 41.

[0222] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0223] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 42.

[0224] In some embodiments, the therapeutic anti-NMDAR antibody includes a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 42.

[0225] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 42.

[0226] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 insertions, deletions, and / or substitutions relative to SEQ ID NO: 42.

[0227] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 42.

[0228] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 relative to SEQ ID NO: 42.

[0229] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0230] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 53, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 54, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 55, and a light chain variable region (VL) comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 56, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 57, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 58.

[0231] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 51.

[0232] In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 51.

[0233] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 51.

[0234] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 insertions, deletions, and / or substitutions relative to SEQ ID NO: 51.

[0235] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, or up to 13 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 51.

[0236] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 relative to SEQ ID NO: 51.

[0237] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0238] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 52.

[0239] In some embodiments, the therapeutic anti-NMDAR antibody includes a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 52.

[0240] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 52.

[0241] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 insertions, deletions, and / or substitutions relative to SEQ ID NO: 52.

[0242] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 52.

[0243] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 relative to SEQ ID NO: 52.

[0244] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0245] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 63, a heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 64, and a heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 65, and a light chain variable region (VL) comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 66, a light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 67, and a light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 68.

[0246] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 61.

[0247] In some embodiments, the therapeutic anti-NMDAR antibody includes a heavy chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 61.

[0248] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region, which includes an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 61.

[0249] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 insertions, deletions, and / or substitutions relative to SEQ ID NO: 61.

[0250] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, or up to 13 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 61.

[0251] In some embodiments, the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 relative to SEQ ID NO: 61.

[0252] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0253] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region which includes an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity or similarity to SEQ ID NO: 62.

[0254] In some embodiments, the therapeutic anti-NMDAR antibody includes a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 62.

[0255] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region, the light chain immunoglobulin variable region comprising an amino acid sequence having one or more amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 62.

[0256] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 insertions, deletions, and / or substitutions relative to SEQ ID NO: 62.

[0257] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 amino acid insertions, deletions, and / or substitutions relative to SEQ ID NO: 62.

[0258] In some embodiments, the therapeutic anti-NMDAR antibody comprises a light chain immunoglobulin variable region comprising an amino acid sequence including insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 relative to SEQ ID NO: 62.

[0259] One or more insertions, deletions, and / or substitutions can be at the N-terminus, C-terminus, within an amino acid sequence, or in combination thereof. One or more insertions can be consecutive, discontinuous, or in combination thereof.

[0260] In some embodiments, a method for detecting pathological anti-NMDAR autoantibodies in a biological sample of an individual further includes the step of quantifying the level of anti-NMDAR autoantibodies in the biological sample.

[0261] In some embodiments, the levels of pathological anti-NMDAR autoantibodies in individual biological samples are at concentrations of up to approximately 500 nμ, up to approximately 400 ng / ml, up to approximately 300 ng / ml, up to approximately 200 ng / ml, up to approximately 100 ng / ml, up to approximately 90 ng / ml, up to approximately 80 ng / ml, up to approximately 70 ng / ml, up to approximately 60 ng / ml, up to approximately 50 ng / ml, up to approximately 40 ng / ml, up to approximately 30 ng / ml, up to approximately 20 ng / ml, up to approximately 10 ng / ml, up to approximately 5 ng / ml, up to approximately 2 ng / ml, or up to approximately 1 ng / ml, or higher.

[0262] In some embodiments, the level of pathological anti-NMDAR autoantibodies in the individual's biological sample is at a concentration of at least about 500 ng / ml, at least about 400 ng / ml, at least about 300 ng / ml, at least about 200 ng / ml, at least about 100 ng / ml, at least about 90 ng / ml, at least about 80 ng / ml, at least about 70 ng / ml, at least about 60 ng / ml, at least about 50 ng / ml, at least about 40 ng / ml, at least about 30 ng / ml, at least about 20 ng / ml, at least about 10 ng / ml, at least about 5 ng / ml, at least about 2 ng / ml, or at least about 1 ng / ml.

[0263] In some embodiments, the levels of pathological anti-NMDAR autoantibodies in individual biological samples are at concentrations of approximately 500 ng / ml or less, approximately 400 ng / ml, approximately 300 ng / ml, approximately 200 ng / ml, approximately 100 ng / ml, approximately 90 ng / ml, approximately 80 ng / ml, approximately 70 ng / ml, approximately 60 ng / ml, approximately 50 ng / ml, approximately 40 ng / ml, approximately 30 ng / ml, approximately 20 ng / ml, approximately 10 ng / ml, approximately 5 ng / ml, approximately 2 ng / ml, or approximately 1 ng / ml.

[0264] In some embodiments, the levels of pathological anti-NMDAR autoantibodies in individual biological samples range from 1-500 ng / ml, 2-500 ng / ml, 5-500 ng / ml, 10-500 ng / ml, 20-500 ng / ml, 30-500 ng / ml, 40-500 ng / ml, 50-500 ng / ml, 60-500 ng / ml, 70-500 ng / ml, 80-500 ng / ml, 90-500 ng / ml, 100-500 ng / ml, 200-500 ng / ml, 300-500 ng / ml, 400-500 ng / ml, 1-400 ng / ml, 2-400 ng / ml, 5-400 ng / ml, and 10-400 ng / ml. 20~400ng / ml, 30~400ng / ml, 40~400ng / ml, 50~400ng / ml, 60~400ng / ml, 70~400ng / ml, 80~400ng / ml, 90~400ng / m l, 100~400ng / ml, 200~400ng / ml, 300~400ng / ml, 1~300ng / ml, 2~300ng / ml, 5~300ng / ml, 10~300ng / ml, 20~300ng / ml, 30~300ng / ml, 40~300ng / ml, 50~300ng / ml, 60~300ng / ml, 70~300ng / ml, 80~300ng / ml, 90~300ng / ml, 100~30 0ng / ml, 200~300ng / ml, 1~200ng / ml, 2~200ng / ml, 5~200ng / ml, 10~200ng / ml, 20~200ng / ml, 30~200ng / ml, 40~200 ng / ml, 50~200ng / ml, 60~200ng / ml, 70~200ng / ml, 80~200ng / ml, 90~200ng / ml, 100~200ng / ml, 1~100ng / ml, 2~100ng / ml, 5~100n g / ml, 10~100ng / ml, 20~100ng / ml, 30~100ng / ml, 40~100ng / ml, 50~100ng / ml, 60~100ng / ml, 70~100ng / ml, 80~100ng / ml, 90~100 ng / ml,1~90ng / ml, 2~90ng / ml, 5~90ng / ml, 10~90ng / ml, 20~90ng / ml, 30~90ng / ml, 40~90ng / ml, 50~90ng / ml, 60~90ng / ml, 70 ~90ng / ml, 80~90ng / ml, 1~80ng / ml, 2~80ng / ml, 5~80ng / ml, 10~80ng / ml, 20~80ng / ml, 30~80ng / ml, 40~80ng / ml, 50~80 ng / ml,60~80ng / ml,70~80ng / ml,1~70ng / ml,2~70ng / ml,5~70ng / ml,10~70ng / ml,20~70ng / ml,30~70ng / ml,40~70ng / ml,50~70ng / ml,60~70ng / ml,1~6 0ng / ml, 2~60ng / ml, 5~60ng / ml, 10~60ng / ml, 20~60ng / ml, 30~60ng / ml, 40~60ng / ml, 50~60ng / ml, 1~50ng / ml, 2~50ng / ml, 5~50ng / ml, 10~50ng / ml, 20~5 The concentration is in the range of 0 ng / ml, 30-50 ng / ml, 40-50 ng / ml, 1-40 ng / ml, 2-40 ng / ml, 5-40 ng / ml, 10-40 ng / ml, 20-40 ng / ml, 30-40 ng / ml, 1-30 ng / ml, 2-30 ng / ml, 5-30 ng / ml, 10-30 ng / ml, 20-30 ng / ml, 1-20 ng / ml, 2-20 ng / ml, 5-20 ng / ml, 10-20 ng / ml, 1-10 ng / ml, 2-10 ng / ml, 5-10 ng / ml, 1-5 ng / ml, 2-5 ng / ml, or 1-2 ng / ml.

[0265] In some embodiments, a method for detecting pathological anti-NMDAR autoantibodies in a biological sample of an individual further includes the step of generating a report that suggests the presence or level of anti-NMDAR antibodies in the biological sample.

[0266] In some embodiments, the report includes the presence or level of anti-NMDAR autoantibodies, an analysis of diseases associated with anti-NMDAR autoantibodies, or a recommendation for treatment with a therapeutic agent. In some embodiments, the report includes the presence or level of anti-NMDAR autoantibodies. In some embodiments, the report includes an analysis of diseases associated with anti-NMDAR autoantibodies disclosed herein. In some embodiments, the report includes a recommendation for treatment with a therapeutic agent such as ART5803. In some embodiments, the report is on a physical medium such as paper, or stored and displayed on a computer, phone, or tablet. In some embodiments, the report is sent to a healthcare provider.

[0267] Those skilled in the art will understand that not all individuals will respond equally to the administered treatment, but nevertheless, these individuals are considered to be deserving of treatment.

[0268] In some embodiments, the antibody can be administered to a subject in need by any route suitable for the administration of the antibody-containing pharmaceutical composition, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously. In some embodiments, the antibody is administered intratumorally. In some embodiments, the antibody is administered according to a preferred administration schedule, such as weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once a month. In some embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective dose. In one embodiment, therapeutically acceptable amounts are 0.1-100 mg / kg, 0.1-90 mg / kg, 0.1-80 mg / kg, 0.1-70 mg / kg, 0.1-60 mg / kg, 0.1-50 mg / kg, 0.1-40 mg / kg, 0.1-30 mg / kg, 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, and 0.1 ~1mg / kg, 0.1~0.5mg / kg, 0.5~100mg / kg, 0.5~90mg / kg, 0.5~80mg / kg, 0.5~70mg / kg, 0.5~60m g / kg, 0.5~50mg / kg, 0.5~40mg / kg, 0.5~30mg / kg, 0.5~20mg / kg, 0.5~10mg / kg, 0.5~5mg / kg, 0.5~1mg / kg, 1~100mg / kg, 1~90mg / kg, 1~80mg / kg, 1~70mg / kg, 1~60mg / kg, 1~50mg / kg, 1~40mg / kg, 1~30mg / kg, 1 ~20mg / kg, 1~10mg / kg, 1~5mg / kg, 5~100mg / kg, 5~90mg / kg, 5~80mg / kg, 5~70mg / kg, 5~60mg / kg, 5~50mg / kg, 5~4 0mg / kg, 5~30mg / kg, 5~20mg / kg, 5~10mg / kg, 10~100mg / kg, 10~90mg / kg, 10~80mg / kg, 10~70mg / kg, 10~60mg / k g, 10~50mg / kg, 10~40mg / kg, 10~30mg / kg, 10~20mg / kg, 20~100mg / kg, 20~90mg / kg, 20~80mg / kg, 20~70mg / kg, 2 0~60mg / kg, 20~50mg / kg, 20~40mg / kg, 20~30mg / kg, 30~100mg / kg, 30~90mg / kg, 30~80mg / kg, 30~70mg / kg, 30~ 60mg / kg, 30~50mg / kg, 30~40mg / kg, 40~100mg / kg, 40~90mg / kg, 40~80mg / kg, 40~70mg / kg, 40~60mg / kg, 40~50m The range is g / kg, 50-100 mg / kg, 50-90 mg / kg, 50-80 mg / kg, 50-70 mg / kg, 50-60 mg / kg, 60-100 mg / kg, 60-90 mg / kg, 60-80 mg / kg, 60-70 mg / kg, 70-100 mg / kg, 70-90 mg / kg, 70-80 mg / kg, 80-100 mg / kg, 80-90 mg / kg, or 90-100 mg / kg. In some embodiments, the therapeutically acceptable amount is 1 mg / kg to 100 mg / kg. In some embodiments, the therapeutically acceptable amount is 2 mg / kg to 50 mg / kg. In some embodiments, the therapeutically acceptable amount is 2 mg / kg to 30 mg / kg. In some embodiments, the therapeutically acceptable amount is 2 mg / kg to 20 mg / kg. A therapeutically effective dose is an amount sufficient to improve one or more symptoms associated with the disease or distress being treated.

[0269] pharmaceutically acceptable excipients, carriers, and diluents In some embodiments, the anti-NMDAR antibody of the present disclosure is contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. The pharmaceutically acceptable excipients, carriers, and diluents may be included to increase the shelf life, stability, or dosability of the antibody. Examples of such compounds include salts, pH buffers, surfactants, anticoagulants, and preservatives. In some embodiments, the antibody of the present disclosure is administered suspended in a sterile solution. In some embodiments, the solution contains about 0.9% NaCl. In some embodiments, the solution contains about 5.0% dextrose. In one embodiment, the solution further comprises buffers, e.g., acetates, citrates, histidines, succinates, phosphates, bicarbonates, and hydroxymethylaminomethane (Tris); surfactants, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyols / disaccharides / polysaccharides, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, e.g., glycine or arginine; antioxidants, e.g., ascorbic acid, methionine; or chelating agents, e.g., one or more of EDTA or EGTA.

[0270] In some embodiments, the antibodies of this disclosure may be shipped / stored, lyophilized, and reconstituted before administration. In some embodiments, the lyophilized antibody formulation may contain a volume extender such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation may be contained in a vial made of glass or other suitable non-reactive material. The antibodies, if formulated, may be buffered at a specific pH, generally below 7.0, whether or not they are reconstituted. In some embodiments, the above pH may be between 4.5 and 7.0, between 4.5 and 6.5, between 4.5 and 6.0, between 4.5 and 5.5, between 4.5 and 5.0, or between 5.0 and 6.0.

[0271] This specification also describes kits comprising one or more of the antibodies described herein in a suitable container, and one or more additional components selected from instructions for use, diluents, excipients, carriers, and administration devices.

[0272] In some embodiments, a method for preparing an anti-NMDAR receptor encephalitis treatment agent is described herein, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the Disclosure. In some embodiments, a method for preparing an anti-NMDAR receptor encephalitis treatment agent for storage or transport is described herein, comprising the step of lyophilizing one or more antibodies of the Disclosure. [Examples]

[0273] The following exemplary examples are representative of the embodiments of compositions and methods described herein and are not intended to limit them in any way.

[0274] Example 1: NTD-NR1, non-randomly coated onto a solid support, enables robust detection of pathogenic antibodies in ELISA assays. Previous studies have shown that the N-terminal domain (NTD) of the NR1 subunit of NMDAR is an autoantibody binding target in patients with anti-NMDAR encephalitis. Pathogenic anti-NMDAR NR1 autoantibodies recognize a selective epitope in the NTD of the NR1 subunit, inducing crosslinking of NMDARs and their internal translocation, resulting in a hypo-NMDAR status. Previous studies identified the epitope sequence in the NTD of the NR1 subunit targeted by the pathogenic autoantibody 102 Ab. The one-arm antibody ART5803 was generated to block the binding of the pathogenic autoantibody 102 Ab to the NMDAR subunit NR1. ART5803 binds to the same epitope as 102 Ab.

[0275] Since pathogenic autoantibodies bind to a limited number of epitopes in the NTD of NMDAR NR1, the orientation of the NTD of NR1 affects the binding affinity of pathogenic autoantibodies. To test whether the orientation of the NTD of NR1 affects the binding affinity of pathogenic autoantibodies, 96-well nickel plates were coated with 200 ng / well of NTD-NR1-His-Avi, and binding to the pathogenic monoclonal autoantibody 102 Ab (with VH of SEQ ID NO: 79 and VL of SEQ ID NO: 80) and the therapeutic antibody ART5803 was evaluated by ELISA assay. Randomly coated NTD-NR1-His-Avi were used as controls. All experiments were performed in assay buffer.

[0276] Specifically, in randomly coated NTD-NR1-His-Avi plates, maxisorp plates (ThermoFisher, #436110) were randomly coated with 8×His-tagged NR1 (disclosed as SEQ ID NO: 70, "8×His") diluted to 2 μg / mL in 1×TBS, added at 100 μL per well, and incubated overnight at 4°C. The plates were then washed five times with 200 μL of 1x TBST (Biotek EL 406), and all subsequent washing steps were performed similarly. Undiluted Blocking One (Nacalai Tesque, #03953-95) was added at 200 μL per well and incubated at room temperature for 1 hour. All subsequent incubations were performed at room temperature. The plates were washed, and antibodies serially diluted in 5% BO / 1x TBS were added at 100 μL per well and incubated for 2 hours. The plates were washed, and 10 μL of goat anti-human IgG HRP conjugate (Bethyl, #A80-140P) in a 1:15,000 ratio in 5% BO / 1×TBS was added to the plates. After washing the plates, BM Chemiluminescence ELISA Substrate (Roche, #11582950001) was prepared according to the manufacturer's instructions and added to the plates at a rate of 100 μL per well. After a 10-minute incubation, chemiluminescence measurements were read using a PerkinElmer Multimode Plate Reader Victor X5. The data were analyzed using a GraphPad Prism 9.

[0277] In non-randomly coated NTD-NR1-His-Avi plates, Pierce Nickel Coated Plates (ThermoFisher #15241) were pre-washed in 200 μL of 1x TBS, non-randomly coated with 8×His-tagged NR1 (disclosed as "8×His" under SEQ ID NO: 70) diluted to 2 μL in 1x TBS, added at 100 μL per well, and incubated overnight at 4°C. The plates were washed five times with 200 μL of 1x TBS (Biotek EL 406), and all subsequent washing steps were performed similarly. After washing the plates, serially diluted antibodies in 5% Bocking One (BO; Nacalai Tesque, #03953-95) / 1x TBS were added at 100 μL per well and incubated for 2 hours. All subsequent incubations were performed at room temperature. The plates were washed, and 100 μL of goat anti-human IgG HRP conjugate (Bethyl, #A80-140P), diluted to 1:15,000 in 5% BO / 1x TBS, was added to the plates. After washing the plates, BM Chemiluminescence ELISA Substrate (Roche, #11582950001) was prepared according to the manufacturer's instructions and added to the plates at a rate of 100 μL per well. After a 10-minute incubation, chemiluminescence measurements were read using a PerkinElmer Multimode Plate Reader Victor X5. The data were analyzed using a GraphPad Prism 9.

[0278] EC of ART5803 and 102 Ab in randomly and non-randomly coated NTD-NR1 50The results are shown in Table 1. ART5803 showed a strong signal in both randomly coated and non-randomly coated NTD-NR1 plates (Figures 2A and 2B). 102 Ab showed significantly lower binding affinity in randomly coated NTD-NR1 plates compared to ART5803 (Figure 2A). However, the signal for 102 Ab improved in non-randomly coated NTD-NR1 plates, becoming similar to the signal for ART5803 (Figure 2B). This result suggests that non-randomly coated NTD-NR1 plates increased the sensitivity of the ELISA for detecting the pathogenic autoantibody 102 Ab. While we do not wish to be bound by any particular theory, the improved sensitivity may be due to the orientation of the NTD-NR1 epitope, which favorably stabilizes the binding of 102 Ab to NTD-NR1 (Figures 1A and 1B).

[0279] [Table 1]

[0280] The Opentrons OT-2 automated liquid handler and Opentrons Protocol Designer (v7.0) were used according to the protocol unless otherwise noted. Prior to coating, Pierce Neutravidin Coated White 384-well plates (ThermoFisher, Ref#15401) were first blocked with 3% Pure BSA IgG-Free (Jackson ImmunoResearch, Ref#001-000-162) in 100 μL of 1x TBS (20X TBS, ThermoFisher, Ref#28358) per well and incubated at room temperature for 1 hour. The plates were then washed three times with 100 μL of 1x TBST (20X TBST, ThermoFisher, Ref#PI28360) using a plate washer (Biotek, Model#EL 406). The plates were coated with 50 μL / well of biotinylated His-Avi-tagged human NR1 in 5 μg / mL 1x TBS. 50 μL / well of 1x TBS was added to the uncoated control wells, followed by centrifugation at 1,200 RPM for 2 minutes (Eppendorf, Model #5910RI), and incubation at 4°C overnight. The plates were then washed five times with 100 μL of 1x TBST using a plate washer. All subsequent washing steps were performed similarly. The plates were again blocked with 100 μL / well of 3% Pure BSA in 1x TBS and incubated at room temperature for 1 hour.Patient-derived anti-NMDA receptor autoantibodies (102Ab, 218Ab, 168Ab, 124Ab) were first manually diluted to 1.0 mg / mL in PBS, pH 7.4 (ThermoFisher, Ref#10010-023), and then triple-diluted in OT-2 on a 96-well Deepwell PP Nunc Plate (Fisher Scientific, Cat#260251) with 50% Blocking One (BO; Nacalai Tesque, Ref#03953-95) / 1x TBS / 10% healthy human pooled serum (Cosmo Bio, Cat#KOJ-12181201C, Lot#20090730) or CSF (BIO IVT, Cat#12102021-CSF-20-2001, Lot#102021-CSF-20). After blocking for 1 hour, the plate was washed, and serially diluted autoantibodies were added to the designated wells at 50 μL / well. The plate was then centrifuged at 1,200 RPM for 2 minutes and incubated at room temperature for 2 hours. All subsequent incubations were also performed at room temperature. After 2 hours of incubation, the plate was washed, and 50 μL of 1:15,000 dilution of goat anti-human IgG HRP conjugate (Bethyl, #A80-140P) in 10% BO / 1x TBS was added. The plate was centrifuged at 1200 RPM for 2 minutes and incubated for a further 2 hours. After incubation, the plate was washed, and BM Chemiluminescence ELISA Substrate (POD, Roche, #11582950001) was prepared according to the manufacturer's instructions and then added to the plate at 50 μL / well. After a 10-minute incubation, chemiluminescence measurements were read using a PerkinElmer Multimode Plate Reader Victor X5, and the data was analyzed using GraphPad Prism (v1 0.0.2).

[0281] Example 2A: NTD-NR1, non-randomly coated onto a solid support, enables robust detection of weak-affinity pathogenic antibodies in an ELISA assay. Because the binding affinity of 102 Ab was improved in non-randomly coated NTD-NR1 plates, three weaker affinity pathogenic monoclonal autoantibodies were tested in a non-randomly coated NTD-NR1 ELISA system using randomly coated NTD-NR1 as a control. This assay was performed essentially as described in Example 1. All three pathogenic monoclonal autoantibodies showed a nearly undetectable signal when using randomly coated NTD-NR1 plates (Figure 3A). 218 Ab, 168 Ab, and 124 Ab showed improved signals in a non-randomly coated NTD-NR1 plate (Figure 3B). These results suggest that a non-randomly coated NTD-NR1 ELISA system can improve the detection of certain weak affinity NMDAR autoantibodies compared to a randomly coated NTD-NR1 ELISA system.

[0282] Similar non-randomly coated NTD-NR1 ELISA systems were prepared using Pierce Neutravidin Coated White 384-well plates (ThermoFisher, Ref#15401) and biotinylated His-Avi-tagged human NR1. Similar to the nickel-based ELISA system, Pierce Neutravidin Coated detected all tested pathogenic monoclonal autoantibodies (Figures 6A and 6B). Indeed, as shown in Figures 6A and 6B, these results could be obtained using different biological samples, including serum and CSF, respectively. In the avidin-coated system, the avidin-coated biotinylated NTD-NR1 ELISA system showed very strong detection of all tested pathogenic monoclonal autoantibodies and good compatibility with high concentrations of healthy human pooled serum and CSF (tested up to 20%, data not shown).

[0283] Various autoantibodies (e.g., IgG / IgA / IgM) were also evaluated in patient serum using a non-randomly coated NTD-NR1 ELISA system (not shown). Previous data have shown that a randomly coated NTD-NR1 ELISA system can only detect low signals of IgG. In some embodiments, a non-randomly coated NTD-NR1 ELISA system can detect IgG / IgA / IgM in patients with weaker affinity NMDAR autoantibodies.

[0284] Pierce Neutravidin Coated White 384-well plates (ThermoFisher, Ref#15401) were used with biotinylated His-Avi-tagged human NR1 cells, similar to those described in Example 1. Pierce Neutravidin Coated detected all tested IgG, IgA, and IgM (Figures 8A and 8B) (102 Ab). Indeed, as shown in Figures 8A and 8B, these results could be obtained using different biological samples, including serum and CSF, respectively.

[0285] Example 2B: NMDAR endogenization assay of 102 IgG / A / M HEK293 cells, stably transfected with tetracycline-inducible expression of human NMDA receptors (GluN1 / GluN2B), were purchased from Charles River Discovery (Cleveland, Ohio, USA). T-REx-293 cells were purchased from Invitrogen (R71007) and prepared in parallel as a negative control using the same treatment as NMDA receptors expressing HEK. The cells were cultured in collagen-coated culture dishes in induction medium (1.5 × 10⁶) consisting of Neurobasal Medium (Life Technologies, Carlsbad, California, USA) containing 10% dialyzed FBS, 50 U / mL penicillin-streptomycin, 2.0 μg / mL tetracycline, and 0.2 mM memantine. 5Cells were induced by incubation in 100 μL of FACS buffer for 16–24 hours. The cells were then harvested and cultured in 96-well plates with the following antibodies to evaluate their internalization activity: 102 IgG and 102 IgA monomeric antibodies were diluted 3-fold in induction medium to 0.0003–10 μg / mL, while 102 IgA dimers were diluted to 0.001–30 ug / mL, and 102 IgM to 0.003–100 ug / mL. Antibodies were added to cells at 100 μL / well and incubated at 37°C, 5% CO2 for 16–24 hours. From this point onward, all steps were maintained on ice. Washing was performed twice with 100 μL / well of FACS buffer, followed by centrifugation at 1200 rpm for 5 minutes at 4°C. Cells were harvested into 96-well round-bottom plates, centrifuged, and the medium was discarded. Human Fc receptor binding inhibitor (1:50; Invitrogen, 14-0161-73) and Live / Dead fixed violet cell stain (1:666; Invitrogen, L34955) were added at 100 μL / well for 15 minutes, followed by centrifugation and discarding of the supernatant. Cells were stained with 5 μg / ml anti-NMDAR antibody 502Ab for 30 minutes. After washing, cells were stained with APC conjugate goat anti-human IgG (1:100; Jackson ImmunoResearch Inc, West Grove, Pennsylvania, USA) for 30 minutes, followed by washing and resuspending in 4% PFA in 100 μL / well PBS. APC signal detection was performed using Novocyte 2060 and NovoExpress v1.5.0 (Agilent Technologies, Santa Clara, California). Flow cytometry data was analyzed using FlowJo v1.7.2 (BD Biosciences, Franklin Lakes, New Jersey, USA). Data were expressed as the mean of two internal replicates. Cell surface NMDA receptor expression levels were calculated after normalizing, with the mean fluorescence intensity (MFI) of 502Ab staining set to 100% surface expression and the mean fluorescence intensity (MFI) of isotype control antibody staining set to 0% surface expression. Data were graphed using GraphPad Prism v1 0.0.2 (GraphPad Software, San Diego, California, USA).

[0286] IgG, IgA, and IgM (102Ab) all exhibit pathogenicity, as they induce significant internal translocation of NMDAR (NR1) in cell-based assays (Figure 7A).

[0287] Example 2C: ART5803 blocks NMDAR internalization induced by 102 IgG / A / M. Using the same NMDAR internalization assay method as described in Example 2B, the following combinations of serially diluted ART5803 (final concentration) and either 102 IgG, IgA dimer, or IgM (final concentration) were co-incubated with cells overnight at 37°C and 5% CO2 for 16–24 hours: ART5803, serially diluted 3-fold to 10–0.001 ug / mL, co-incubated with 10 ug / mL of 102 IgG; ART5803, serially diluted 3-fold to 10–0.001 ug / mL, co-incubated with 3 ug / mL of 102 IgA dimer; and ART5803, serially diluted 3-fold to 100–0.01 ug / mL, co-incubated with 3 ug / mL of 102 IgM. ART503 blocks NMDAR(NR1) internalization induced by IgG, IgA, and IgM (Ab of 102) in cell-based assays (Figures 7B, 7C, and 7D).

[0288] Example 3: ART5803-Fab displaces the bound NMDAR autoantibody from NTD-NR1. When NMDAR autoantibodies bind to the same epitope as ART5803, ART5803 exhibits a stronger affinity for NTD-NR1, suggesting that ART5803 may be able to displace the binding of NMDAR autoantibodies to NTD-NR1. ART5803-Fab was generated and used to test whether NMDAR autoantibodies could be displaced by ART5803-Fab in a non-randomly coated NTD-NR1 ELISA system (Figure 4).

[0289] Specifically, Pierce Nickel Coated Plates (ThermoFisher #15241) were pre-washed with 200 μL 1x TBS, randomly coated with 8x His-tagged NR1 (disclosed as SEQ ID NO: 70) diluted to 2 μL in 1x TBS, added at a rate of 100 μL per well, and incubated overnight at 4°C. The plates were washed five times with 200 μL of 1x TBS (Biotek EL 406), and all subsequent washing steps were performed similarly. ART5803-Fab, serially diluted in 5% Blocking One (BO; Nacalai Tesque, #03953-9) / 1x TBS, was added to the plates at a rate of 50 μL / well and incubated at room temperature for 2 hours. All subsequent incubation steps were also performed at room temperature. Based on previous assessments of the saturation dose of pathogenic monoclonal autoantibodies bound in non-randomized coated NR1 ELISA, pathogenic antibodies were added to plates at 50 μL / well and incubated for 2 hours (final concentrations diluted with 5% BO / 1x TBS were 0.12 ug / mL for 102Ab, 300 ug / mL for 124Ab, 33.33 ug / mL for 168Ab, and 3.7 ug / mL for 218Ab). The plates were washed, and 100 μL of goat anti-human IgG HRP conjugate (Bethyl, #A80-140P) diluted 1:15,000 in 5% BO / 1x TBS was added to the plates. After washing the plates, BM Chemiluminescence ELISA Substrate (Roche, #11582950001) was prepared according to the manufacturer's instructions and added to the plates at 100 μL per well. After a 10-minute incubation, chemiluminescence measurements were read using a PerkinElmer Multimode Plate Reader Victor X5. The data were transformed and normalized using a GraphPad Prism 9 to evaluate the percentage of inhibition of pathogenic antibody binding by ART5803-Fab.

[0290] The binding of all four pathogenic monoclonal autoantibodies (102Ab, 218Ab, 168Ab, and 124Ab) to NTD-NR1 was completely displaced by higher concentrations of ART5803-Fab (Figure 5), demonstrating that ART5803-Fab competes for binding to pathogenic monoclonal antibodies to NTD-NR1. This method can provide information regarding autoantibody epitope binding specificity, pathogenicity, and whether patients with autoantibodies respond to ART5803 treatment.

[0291] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided only as examples. Numerous variations, modifications, and substitutions are now conceivable by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein are available for the practice of the present invention.

[0292] All publications, patent applications, issued patents, and other documents referenced herein are incorporated by reference in such a manner as each individual publication, patent application, issued patent, or other document is specifically and individually incorporated in whole by reference. Definitions contained in the documents incorporated by reference are excluded to the extent that they conflict with the definitions in this disclosure.

[0293] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] Table 2-6

Claims

1. A plurality of N-methyl-D-aspartate receptor (NMDAR) antigens coupled to a solid support, wherein the plurality of NMDAR antigens are coupled non-randomly to the solid support.

2. The plurality of NMDAR antigens according to claim 1, wherein the solid support comprises a plate, a test tube, a microtiter well, beads, a slide, a membrane, fine particles, nanoparticles, or a chip.

3. The plurality of NMDAR antigens according to claim 1 or 2, wherein the solid support comprises plastic, derivatized plastic, polystyrene, polyvinyl chloride, magnetic metal, non-magnetic metal, glass, or silicon.

4. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in any one of sequence numbers 1 to 5.

5. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in SEQ ID NO:

1.

6. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in SEQ ID NO:

2.

7. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in SEQ ID NO:

3.

8. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in SEQ ID NO:

4.

9. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in SEQ ID NO:

5.

10. The plurality of NMDAR antigens according to any one of claims 1 to 3, wherein the plurality of NMDAR antigens include the amino acid sequence shown in SEQ ID NO:

83.

11. The plurality of NMDAR antigens according to any one of claims 1 to 10, wherein the plurality of NMDAR antigens lack an N-terminal signal peptide.

12. The plurality of NMDAR antigens according to any one of claims 1 to 11, wherein the plurality of NMDAR antigens do not include the GluN2A domain, GluN2B domain, GluN2C domain, GluN2D domain, or any combination thereof of the NMDAR.

13. The plurality of NMDAR antigens according to any one of claims 1 to 12, wherein the plurality of NMDAR antigens are covalently coupled to the solid support.

14. The plurality of NMDAR antigens according to any one of claims 1 to 12, wherein the plurality of NMDAR antigens are non-covalently coupled to the solid support.

15. The plurality of NMDAR antigens according to any one of claims 1 to 14, wherein the plurality of NMDAR antigens further comprises an Fc polypeptide.

16. The plurality of NMDAR antigens according to claim 15, wherein the Fc polypeptide is bound to the C-terminus of the plurality of NMDAR antigens.

17. The plurality of NMDAR antigens according to claim 15, wherein the Fc polypeptide is bound to the N-terminus of the plurality of NMDAR antigens.

18. The plurality of NMDAR antigens according to any one of claims 15 to 17, wherein the Fc polypeptide is a mouse Fc polypeptide.

19. The plurality of NMDAR antigens according to any one of claims 1 to 18, wherein the plurality of antigens include affinity tags.

20. The plurality of NMDAR antigens according to claim 19, wherein the affinity tag is coupled to the C-terminus of the plurality of NMDAR antigens.

21. The plurality of NMDAR antigens according to claim 19, wherein the affinity tag is coupled to the N-terminus of the plurality of NMDAR antigens.

22. A plurality of NMDAR antigens according to any one of claims 19 to 21, wherein the affinity tag includes His tag, FLAG tag, c-Myc tag, HA tag, V5 tag, GST tag, MBP tag, CBP tag, CBD tag, Avi- tag, biotinylated Avi- tag, or a combination thereof.

23. A plurality of NMDAR antigens according to any one of claims 19 to 21, wherein the affinity tag includes a biotinylated Avi-tag.

24. The plurality of NMDAR antigens according to claim 23, wherein the biotinylated Avi-tag is coupled to a solid support containing avidin.

25. The plurality of NMDAR antigens according to claim 24, wherein the avidin is a deglycosylated avidin (e.g., neutraavidin or streptavidin).

26. A plurality of NMDAR antigens according to any one of claims 19 to 21, wherein the affinity tag includes a His tag.

27. A plurality of NMDAR antigens according to claim 22 or 26, wherein the His tag is non-covalently coupled to a nickel-containing solid support.

28. The plurality of NMDAR antigens according to claim 27, wherein the nickel-containing solid support is a well of an enzyme-linked immunosorbent assay (ELISA) plate.

29. A plurality of NMDAR antigens according to any one of claims 1 to 28, wherein the individual NMDAR antigens of the plurality of NMDAR antigens bound non-randomly to the solid support are separated by an average distance of 15 nm or less.

30. The plurality of NMDAR antigens according to any one of claims 1 to 29, wherein the plurality of NMDAR antigens are bound to an anti-NMDAR antibody.

31. The plurality of NMDAR antigens according to any one of claims 1 to 29, wherein the plurality of NMDAR antigens are bound to a pathogenic anti-NMDAR antibody.

32. A method for detecting pathological anti-NMDAR antibodies in a biological sample of an individual, comprising the steps of: contacting the biological sample of the individual with a plurality of NMDAR antigens described in any one of claims 1 to 31; and detecting the binding of the pathological anti-NMDAR antibodies in the biological sample to the plurality of NMDAR antigens.

33. The method according to claim 32, wherein the biological sample includes blood, plasma, serum, saliva, cell lysate, lymph, amniotic fluid, cerebrospinal fluid, tears, mucus, urine, sputum, amniotic fluid, or sweat.

34. The method according to claim 32, wherein the biological sample includes blood.

35. The method according to claim 32, wherein the biological sample includes plasma.

36. The method according to claim 32, wherein the biological sample includes serum.

37. The method according to claim 32, wherein the biological sample includes lymph fluid.

38. The method according to claim 32, wherein the biological sample includes cerebrospinal fluid.

39. The method according to claim 32, wherein the biological sample includes amniotic fluid.

40. The method according to any one of claims 32 to 39, wherein the binding of the pathological anti-NMDAR antibody to the plurality of NMDAR antigens is detected by an enzymatic reaction.

41. The method according to any one of claims 32 to 40, wherein the binding of the pathological anti-NMDAR antibody to the plurality of NMDAR antigens is detected by a fluorescence signal.

42. The method according to any one of claims 32 to 41, wherein the binding of the pathological anti-NMDAR antibody to the plurality of NMDAR antigens is detected by immunoassay.

43. The method according to claim 42, wherein the immunoassay comprises ELISA.

44. The method according to claim 43, wherein the ELISA comprises contacting the biological sample of the individual with the plurality of NMDAR antigens to produce a pathological anti-NMDAR antibody / NMDAR antigen complex, and contacting the pathological anti-NMDAR antibody / NMDAR antigen complex with a detection agent that binds to the pathological anti-NMDAR antibody.

45. The method according to claim 44, wherein the detection agent is an anti-isotype antibody.

46. The method according to claim 44 or 45, wherein the detection agent is labeled with a fluorescent compound or an enzyme.

47. The method according to any one of claims 32 to 41, wherein the binding of the pathological anti-NMDAR antibody to the plurality of NMDAR antigens is detected by fluorescence-activated cell sorting (FACS).

48. The method according to any one of claims 32 to 47, wherein the anti-NMDAR antibody present in the biological sample is detected at a concentration between 0.3 ng / mL and 100 μg / mL.

49. The method according to any one of claims 32 to 48, wherein the presence of the aforementioned pathological anti-NMDAR antibody suggests a diagnosis of autoimmune encephalitis, dementia, psychosis, schizophrenia, bipolar disorder, seizures, epilepsy, or depression.

50. The method according to any one of claims 32 to 48, wherein the presence of the aforementioned pathological anti-NMDAR antibody suggests a diagnosis of autoimmune encephalitis.

51. The method according to any one of claims 32 to 50, further comprising the step of quantifying the level of the pathological anti-NMDAR antibody in the biological sample.

52. The method according to any one of claims 32 to 51, further comprising the step of generating a report indicating the presence or level of the anti-NMDAR antibody in the biological sample.

53. The method according to any one of claims 32 to 52, further comprising the step of administering to the individual a therapeutic anti-NMDAR antibody or an anti-NMDAR-binding fragment thereof, which prevents the binding of pathological anti-NMDAR autoantibodies to NMDAR in the individual, based on the detection of the presence of the aforementioned pathological anti-NMDAR antibody.

54. The therapeutic anti-NMDAR antibody is The heavy chain variable region (VH), a) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 13, b) Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 14, and / or c) Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 15 The heavy chain variable region (VH), Light chain variable region (VL), d) Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 16, e) Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 17, and / or f) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 18 Including the light chain variable region (VL) and The method according to claim 53, including the method described in claim 53.

55. The therapeutic anti-NMDAR antibody is The heavy chain variable region (VH), a) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 23, b) Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 24, and / or c) Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 25 The heavy chain variable region (VH), The light chain variable region (VL) is, d) Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 26, e) Light chain complementarity determination region 2 (LCDR2) containing amino acid sequence EDN, and / or f) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 28 Including the light chain variable region (VL) and The method according to claim 53, including the method described in claim 53.

56. The therapeutic anti-NMDAR antibody is The heavy chain variable region (VH), a) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 33, b) Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 34, and / or c) Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 35 The heavy chain variable region (VH), Light chain variable region (VL), d) Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 36, e) Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 37, and / or f) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 38 Including the light chain variable region (VL) and The method according to claim 53, including the method described in claim 53.

57. The therapeutic anti-NMDAR antibody is The heavy chain variable region (VH), a) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 43, b) Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 44, and / or c) Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 45 The heavy chain variable region (VH), Light chain variable region (VL), d) Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 46, e) Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 47, and / or f) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 48 Including the light chain variable region (VL) and The method according to claim 53, including the method described in claim 53.

58. The therapeutic anti-NMDAR antibody is The heavy chain variable region (VH), a) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 53, b) Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 54, and / or c) Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 55 The heavy chain variable region (VH), Light chain variable region (VL), d) Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 56, e) Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 57, and / or f) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 58 Including the light chain variable region (VL) and The method according to claim 53, including the method described in claim 53.

59. The therapeutic anti-NMDAR antibody is The heavy chain variable region (VH), a) Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 63, b) Heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 64, and / or c) Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 65 and The heavy chain variable region (VH), Light chain variable region (VL), d) Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 66, e) Light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 67, and / or f) Light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO: 68 Including the light chain variable region (VL) and The method according to claim 53, including the method described in claim 53.

60. The therapeutic anti-NMDAR antibody is The heavy chain immunoglobulin variable region and the light chain immunoglobulin variable region, a) Sequence ID 11 and Sequence ID 12, b) Sequence IDs 21 and 22, c) Sequence ID 31 and Sequence ID 32, d) Sequence ID 41 and Sequence ID 42, e) Sequence ID 51 and Sequence ID 52, or f) Sequence IDs 61 and 62 The method according to claim 53, comprising a heavy chain immunoglobulin variable region and a light chain immunoglobulin variable region selected from.

61. The method according to claim 53, wherein the therapeutic anti-NMDAR antibody comprises a heavy chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO: 11 and a light chain immunoglobulin variable region containing the amino acid sequence of SEQ ID NO:

12.

62. The method according to claim 53, wherein the therapeutic anti-NMDAR antibody comprises a heavy-chain immunoglobulin variable region having at least 90% sequence identity with respect to the sequence of SEQ ID NO: 11, and a light-chain immunoglobulin variable region having at least 90% sequence identity with respect to the sequence of SEQ ID NO:

12.

63. The method according to any one of claims 53 to 62, wherein the therapeutic anti-NMDAR antibody is a one-arm antibody.

64. The method according to any one of claims 32 to 63, wherein the step of detecting the binding of the pathological anti-NMDAR antibody in the biological sample includes detecting the binding of the IgG isotype of the pathological anti-NMDAR antibody.

65. The method according to any one of claims 32 to 63, wherein the step of detecting the binding of the pathological anti-NMDAR antibody in the biological sample includes detecting the binding of the IgA isotype of the pathological anti-NMDAR antibody.

66. The method according to any one of claims 32 to 63, wherein the step of detecting the binding of the pathological anti-NMDAR antibody in the biological sample includes detecting the binding of the IgM isotype of the pathological anti-NMDAR antibody.

67. The method according to any one of claims 32 to 63, wherein the step of detecting the binding of the pathological anti-NMDAR antibody in the biological sample is not dependent on whether the pathological anti-NMDAR antibody in the biological sample is IgA, IgM, or IgG.

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