Methods and systems for identifying or treating central nervous system disorders

Abstract

Systems and methods for identifying autoantibodies associated with central nervous system or psychiatric diseases or disorders. Also disclosed herein are methods for treating autoantibody-associated central nervous system or psychiatric diseases or disorders.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 369,923, filed July 29, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Autoimmunity results from the body's production of T cells and / or antibodies specific to self-antigens. This immune response can be triggered by viruses, environmental exposures containing pathogenic organisms, or tumor growth. The immune response can become chronic, resulting in a variety of illnesses, including mental and central nervous system (CNS) disorders or diseases caused by the production of autoantibodies. Viral, bacterial, and parasitic infections, particularly toxoplasmosis, are known to increase the risk of schizophrenia and related psychiatric disorders. One potential mechanism for infection-induced CNS disease is molecular mimicry, where proteins from pathogenic organisms have sequences similar to proteins expressed in the human brain, generating cross-reactive autoantibodies. There is a need to identify autoantibodies, related antigenic peptides, and treatments for autoantibody-related diseases. Summary of the Invention

[0003] Provided herein is a method for treating or ameliorating a psychiatric or central nervous system disease or disorder, comprising administering to a subject in need of treatment or amelioration of a psychiatric or central nervous system disease or disorder an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are: (a) a heavy chain CDR1 set forth in SEQ ID NO: 11, a heavy chain CDR2 set forth in SEQ ID NO: 12, a heavy chain CDR3 set forth in SEQ ID NO: 13, a light chain CDR1 set forth in SEQ ID NO: 14, a light chain CDR2 set forth in SEQ ID NO: 15, and / or a light chain CDR3 set forth in SEQ ID NO: 16; (b) a heavy chain CDR1 set forth in SEQ ID NO: 17, a heavy chain CDR2 set forth in SEQ ID NO: 18, a heavy chain CDR3 set forth in SEQ ID NO: 19, a light chain CDR1 set forth in SEQ ID NO: 20, a light chain CDR2 set forth in SEQ ID NO: 21, and / or a light chain CDR3 set forth in SEQ ID NO: 22; (c) a heavy chain CDR1 set forth in SEQ ID NO: 23, a heavy chain CDR2 set forth in SEQ ID NO: 24, a heavy chain CDR3 set forth in SEQ ID NO: 25, a light chain CDR1 set forth in SEQ ID NO: 26, a light chain CDR2 set forth in SEQ ID NO: 27, and / or a light chain CDR3 set forth in SEQ ID NO: 28; (d) a heavy chain CDR1 set forth in SEQ ID NO: 29, a heavy chain CDR2 set forth in SEQ ID NO: 30, a heavy chain CDR3 set forth in SEQ ID NO: 31, a light chain CDR1 set forth in SEQ ID NO: 32, a light chain CDR2 set forth in SEQ ID NO: 33, and / or a light chain CDR3 set forth in SEQ ID NO: 34, or (e) a heavy chain CDR1 set forth in SEQ ID NO: 35, a heavy chain CDR2 set forth in SEQ ID NO: 36, a heavy chain CDR3 set forth in SEQ ID NO: 37, a light chain CDR1 set forth in SEQ ID NO: 38, a light chain CDR2 set forth in SEQ ID NO: 39, and / or a light chain CDR3 set forth in SEQ ID NO: 40. The heavy chain variable region may comprise an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region may comprise an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, or 10. The heavy chain variable region may comprise an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region may comprise an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, or 10. The heavy chain variable region may comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region may comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, or 10. The heavy chain variable region may comprise an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region may comprise an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, or 10. The heavy chain variable region may comprise an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region may comprise an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, or 10. The heavy chain variable region may comprise an amino acid sequence identical to any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region may comprise an amino acid sequence identical to any one of SEQ ID NOs: 2, 4, 6, 8, or 10.

[0004] The psychiatric or central nervous system disease or disorder may be an autoantibody-related disease.The psychiatric or central nervous system disease or disorder may be schizophrenia.The psychiatric or central nervous system disease or disorder may be psychosis, bipolar disorder, depression, epilepsy, and dementia.In some embodiments, the psychiatric or central nervous system disease or disorder is not encephalitis.

[0005] The antibody or antigen-binding fragment thereof may bind to an antigen expressed by neurons and / or glia of the subject. The antibody or antigen-binding fragment thereof may bind to the N-methyl-D-aspartate (NMDA) receptor of the subject. The antibody or antigen-binding fragment thereof may bind to the NMDAR1 (NR1) subunit of the NMDA receptor. The antibody or antigen-binding fragment thereof may bind to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41). The antibody or antigen-binding fragment thereof may bind to at least one amino acid of NR1 consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41).

[0006] Disclosed herein is a method for identifying an antibody or antigen-binding fragment thereof for use in treating a mental or central nervous system disease or disorder, the method comprising identifying the antibody or antigen-binding fragment thereof having the following property: specific binding to the epitope LQNRKLV (SEQ ID NO: 41) of the NR1 subunit of the NMDA receptor. The antibody or antigen-binding fragment thereof can inhibit binding of autoantibodies that bind to the NMDA receptor. The antibody or antigen-binding fragment thereof has a K of less than 1 mM. D The antibody or antigen-binding fragment thereof may comprise a human antibody. The antibody or antigen-binding fragment thereof may be humanized.

[0007] Disclosed herein is a method for treating or ameliorating a mental or central nervous system disease or disorder, comprising identifying a subject as having been exposed to a pathogenic organism, wherein the identifying step comprises identifying an antibody that binds to an immunogenic epitope of the pathogenic organism. The method may further comprise identifying the subject as having one or more symptoms of a mental or central nervous system disease or disorder. The method may further comprise outputting a report identifying the subject as having a high risk of N-methyl-D-aspartate (NMDA) receptor dysfunction. The mental or central nervous system disease or disorder may be anti-NMDAR encephalitis, schizophrenia, psychosis, bipolar disorder, depression, epilepsy, or dementia. The mental or central nervous system disease or disorder is not encephalitis. The pathogenic organism may be of the genus Toxoplasma, Paramecium, Campylobacter, Enterococcus, Peptoniphilus, Paenalcaligenes, Pseudomonas, Burkholderia, Chromobacterium, Acinetobacter, Paenibacillus, Escherichia, or Nocardia. The pathogenic organism may be any one or more of the pathogenic organisms listed in Table 3. The one or more symptoms may include delusional thought, seizures, speech disorders, difficulty with movement, or aggression.

[0008] Disclosed is a method for identifying a subject as being at risk for an autoantibody-related disease, comprising obtaining a biological sample from the subject, assaying the biological sample for antibodies that bind to the amino acid sequence LQNRKLV (SEQ ID NO: 41), and optionally outputting a report or identifying the subject as being at high or low risk for the autoantibody-related disease. The biological sample may include blood, sweat, saliva, cerebrospinal fluid (CSF), amniotic fluid, or mucus. The subject may be pregnant. The risk of the autoantibody-related disease may be associated with the subject's fetus. The method may further include treating the subject for the autoantibody-related disease. The treating step may include administering an FcRn receptor blocking compound to the subject. The FcRn receptor blocking compound may be a polypeptide. The FcRn receptor blocking compound may be an antibody or an antigen-binding fragment thereof. The FcRn receptor blocking compound may be selected from the group consisting of rozanolixizumab, SYNT001, M281, Argx-113, HL161-11G, HL161-11H, HL161-1A, DX-2504, DX-2507, ABY039, IMVT-1401 / RVT1401, and combinations thereof.

[0009] Disclosed herein is a method for treating or ameliorating a mental or central nervous system disease or disorder, comprising administering to a subject in need of treatment or amelioration of the mental or central nervous system disease or disorder an antibody or fragment that binds to the N-methyl-D-aspartate (NMDA) receptor of the subject. The antibody or antigen-binding fragment thereof can bind to the NMDAR1 (NR1) subunit of the NMDA receptor. The antibody or antigen-binding fragment thereof can bind to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41). The antibody or antigen-binding fragment thereof can bind to at least one amino acid of NR1 consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41).

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0011] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Figure 1] 1 illustrates the binding of the pathogenic autoantibody fragments described herein to the NR1 subunit of the NMDA receptor. [Figure 2] 1 illustrates exemplary residues of the antibody fragments described herein that interact with amino acid residues of the NR1 subunit of the NMDA receptor. [Figure 3] 1 illustrates a hydrogen-deuterium exchange analysis of the binding of an antibody described herein to the N-terminus of the NR1 subunit of the NMDA receptor. [Figure 4] 1 illustrates the predicted epitope structure of the NR1 subunit of the NMDA receptor. [Figure 5] 1 illustrates HDX mass spectrometry of anti-NR1 subunit and therapeutic antibodies. [Figure 6] 1 illustrates HDX mass spectrometry of anti-NR1 subunit and pathogenic autoantibodies. [Figure 7] The entire sequence of the Toxoplasma gondii internal kinesin motor domain protein is illustrated. [Figure 8A] Illustrated are surface probability plots and associated antigenic indices for peptide sequences of core epitopes of the NR1 subunit of the NMDA receptor (8A) compared to the kinesin protein of Toxoplasma gondii (8B). [Figure 8B]Illustrated are surface probability plots and associated antigenic indices for peptide sequences of core epitopes of the NR1 subunit of the NMDA receptor (8A) compared to the kinesin protein of Toxoplasma gondii (8B). DETAILED DESCRIPTION OF THE INVENTION

[0012] In the following description, certain details are set forth to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will understand that the provided embodiments may be practiced without these details. Unless the context otherwise requires, throughout the specification and the following claims, the word "comprise" and variations thereof, such as "comprises" or "comprising," are to be construed in their open and inclusive sense, i.e., "including, but not limited to," unless the context otherwise requires. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0013] Although certain embodiments and examples are disclosed below, the subject matter of the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or alternative uses, as well as modifications and equivalents thereof. Accordingly, the claims appended hereto are not limited by any of the specific embodiments described below. For example, in any method or process disclosed herein, the acts or operations of the method or process may be performed in any suitable order and are not necessarily limited to any particular order disclosed. Although various operations may be described sequentially as multiple separate operations in a manner that may be useful for understanding a particular embodiment, the order of description should not be construed to imply that these operations are order-dependent. Furthermore, the structures, systems, and / or devices described herein may be implemented as integrated components or as separate components.

[0014] For purposes of comparing various embodiments, certain aspects and advantages of these embodiments are described. Not all such aspects or advantages are necessarily achieved by any particular embodiment. Thus, for example, various embodiments may be implemented in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other aspects or advantages that may also be taught or suggested herein.

[0015] As used in this specification and claims, including in the examples, unless expressly stated otherwise, all numbers can be read as if preceded by the term "about" or "approximately," even if that term does not explicitly appear. As used herein, the term "about" refers to an amount that is 10% or less closer to the stated amount. In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the figures, like symbols typically identify like components unless the content dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein. The phrase "about" or "approximately" can be used when describing a size and / or location to indicate that the stated value and / or location is within a reasonably expected range of values and / or locations. For example, a numerical value can have a value of ±0.1% of the stated value (or range of values), ±1% of the stated value (or range of values), ±2% of the stated value (or range of values), ±5% of the stated value (or range of values), ±10% of the stated value (or range of values), etc. Any numerical value given herein should also be understood to include about or approximately that value, unless the context dictates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical ranges described herein are intended to include all subranges encompassed therein. When a value is disclosed, it is understood that "less than or equal to" the value, "greater than or equal to" the value, and possible ranges between the values are also disclosed, as appropriately understood by those of skill in the art. For example, if a value "X" is disclosed the disclosure also includes "greater than or equal to X" as well as "less than or equal to X" (eg, where X is a number).It is also understood that throughout this application, data is provided in many different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that values greater than, greater than, less than, less than, equal to, and between 10 and 15 are considered to be disclosed. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0016] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," mean that various components may be used jointly in methods and articles (e.g., compositions and apparatuses that include devices and methods). For example, the term "comprising" will be understood to mean the inclusion of any stated element or step, but not the exclusion of any other elements or steps.

[0017] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease that the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

[0018] Among the antibodies provided are monoclonal antibodies and antibody fragments. Antibodies include antibody-containing molecules such as antibody conjugates and chimeric molecules. Thus, antibodies include full-length and native antibodies, as well as any specific binding portion thereof, including those of any of the immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including, but not limited to, Fab, F(ab')2, Fv, and scFv (single-chain or related units). Monoclonal antibodies are generally one in a substantially homogeneous antibody composition; therefore, all individual antibodies contained within a monoclonal antibody composition are identical except for possible naturally occurring mutations, which may be present in minor amounts. Monoclonal antibodies may contain a human IgG1 constant region. Monoclonal antibodies may contain a human IgG4 constant region.

[0019] The term "antibody" herein is used in the broadest sense and includes monoclonal antibodies, as well as intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term antibody encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies; multispecific antibodies, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies; tandem di-scFv; tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term antibody also encompasses whole or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0020] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids in an 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 has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).The exact amino acid sequence boundaries of a given CDR or FR can be determined using the methods of Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland ("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), 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 scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0021] The boundaries of a given CDR or FR may vary depending on the scheme used for identification.For example, Kabat scheme is based on structural alignment, while Chothia scheme is based on structural information.The numbering of both Kabat scheme and Chothia scheme is based on the most common antibody region sequence length, and insertions and deletions, which are represented by insertion letters such as "30a", occur in some antibodies.The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering.The Contact scheme is based on the analysis of complex crystal structures, and in many respects is similar to the Chothia numbering scheme.

[0022] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and V L ) have a generally similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). To confer antigen-binding specificity, a single V H Domain or V L Furthermore, antibodies that bind to a particular antigen may be obtained by isolating a V domain from the antibody that binds to the antigen. H Domain or V L domains to form complementary V L Domain or V HDomains can be isolated by screening libraries of each (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0023] The specific binding or binding of antibody molecules described herein refers to the binding mediated by one or more CDR portions of antibody.Not all CDRs are necessarily required for specific binding.Specific binding can be demonstrated, for example, by ELISA against the specific target or antigen, which shows significant increase in binding compared with isotype control antibody.

[0024] As used herein, "epitope" refers to the binding determinant of an antibody or fragment described herein that is minimally required for specific binding of the antibody or antigen-binding fragment thereof to the target antigen. When the target antigen is a polypeptide, the epitope may be a continuous or discontinuous epitope. A continuous epitope is formed by a single region of the target antigen, while a discontinuous epitope may be formed from two or more distinct regions. A discontinuous epitope may be formed, for example, when the target antigen adopts a tertiary structure that combines two amino acid sequences to form a tertiary structure to which an antibody binds. When the target antigen is a polypeptide, the epitope is generally multiple 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. An epitope may comprise a contiguous polymer of amino acids, although not all amino acids of the polymer may contact amino acid residues of an antibody. Such non-contacting amino acids may still comprise part of the epitope, as they may be important for the structure and binding of the contacting amino acids. One skilled in the art may determine whether any given antibody binds to the epitope of a reference antibody, for example, by cross-blocking experiments using a reference antibody. In certain embodiments, antibodies that bind to the same epitope as the described antibodies are described herein. In certain embodiments, antibodies that are competitively blocked by the described antibodies are described herein. In certain embodiments, antibodies that compete for binding with the described antibodies are described herein.

[0025] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen that the complete 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 the variable heavy chain region and / or the variable light chain region, such as an scFv.

[0026] Antibody fragments can be produced 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, e.g., a fragment containing a non-naturally occurring configuration, such as two or more antibody regions or chains joined by a synthetic linker, e.g., a polypeptide linker, and / or one that is not produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is an scFv.

[0027] A "humanized" antibody is an antibody 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 contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0028] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence, including a human antibody library. The term human antibody excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, e.g., those in which all or substantially all CDRs are non-human.

[0029] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, that contain antibody coding sequences derived from the human repertoire.

[0030] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and connecting peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, polypeptides can contain modifications relative to the native or native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as by site-directed mutagenesis, or accidental, such as due to mutations of hosts producing the protein or errors due to PCR amplification. In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptides described herein, and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody, and amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0031] The percent (%) sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Suitable parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, 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 submitted to 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 must 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 do not vary.

[0032] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It is 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 equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program, as described in the immediately preceding paragraph.

[0033] In some embodiments, the methods described herein include treating or ameliorating a psychiatric or central nervous system disease or disorder. In some embodiments, the psychiatric or central nervous system disease or disorder is an autoantibody-associated disease. In some embodiments, the psychiatric or central nervous system disease or disorder is a neurodegenerative or cognitive disease or disorder. In some embodiments, the psychiatric or central nervous system disease or disorder may be schizophrenia, psychosis, bipolar disorder, depression, epilepsy, or dementia.

[0034] In some embodiments, the methods described herein include administering to a subject in need thereof an antibody or antigen-binding fragment thereof that binds to neurons or glia. In some embodiments, the antibody or antigen-binding fragment thereof binds to a neuronal receptor or glial receptor. In some embodiments, the neuronal receptor or glial receptor is a ligand-gated cation channel. In some embodiments, the ligand-gated cation channel is activated by glutamate. In some embodiments, the neuronal receptor is a G protein-coupled ionotropic glutamate receptor. In some embodiments, the antibody or antigen-binding fragment thereof binds to the NMDAR1 (NR1) subunit of the NMDA receptor. In some embodiments, the antibody or antigen-binding fragment thereof may bind to at least one amino acid of the NR1 epitope consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least two amino acids of the NR1 epitope consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41).

[0035] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least one amino acid of NR1 consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41).

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 1, 3, 5, 7, or 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having CDR regions having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 11, 12, 13, 17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36, or 37.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 2, 4, 6, 8, or 10. In some embodiments, the antibody or fragment thereof comprises a light chain having a CDR region having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 14, 15, 16, 20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39, or 40.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a heavy chain CDR1 set forth in SEQ ID NO: 11, a heavy chain CDR2 set forth in SEQ ID NO: 12, a heavy chain CDR3 set forth in SEQ ID NO: 13, a light chain CDR1 set forth in SEQ ID NO: 14, a light chain CDR2 set forth in SEQ ID NO: 15, and / or a light chain CDR3 set forth in SEQ ID NO: 16.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a heavy chain CDR1 set forth in SEQ ID NO: 17, a heavy chain CDR2 set forth in SEQ ID NO: 18, a heavy chain CDR3 set forth in SEQ ID NO: 19, a light chain CDR1 set forth in SEQ ID NO: 20, a light chain CDR2 set forth in SEQ ID NO: 21, and / or a light chain CDR3 set forth in SEQ ID NO: 22.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a heavy chain CDR1 set forth in SEQ ID NO: 23, a heavy chain CDR2 set forth in SEQ ID NO: 24, a heavy chain CDR3 set forth in SEQ ID NO: 25, a light chain CDR1 set forth in SEQ ID NO: 26, a light chain CDR2 set forth in SEQ ID NO: 27, and / or a light chain CDR3 set forth in SEQ ID NO: 28.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a heavy chain CDR1 set forth in SEQ ID NO: 29, a heavy chain CDR2 set forth in SEQ ID NO: 30, a heavy chain CDR3 set forth in SEQ ID NO: 31, a light chain CDR1 set forth in SEQ ID NO: 32, a light chain CDR2 set forth in SEQ ID NO: 33, and / or a light chain CDR3 set forth in SEQ ID NO: 34.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a heavy chain CDR1 set forth in SEQ ID NO: 35, a heavy chain CDR2 set forth in SEQ ID NO: 36, a heavy chain CDR3 set forth in SEQ ID NO: 37, a light chain CDR1 set forth in SEQ ID NO: 38, a light chain CDR2 set forth in SEQ ID NO: 39, and / or a light chain CDR3 set forth in SEQ ID NO: 40.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO: 1, 3, 5, 7, or 9, and a light chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO: 2, 4, 6, 8, or 10.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:1 and a light chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:2.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:3 and a light chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:4.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:5 and a light chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:6.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO: 7 or 9, and a light chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO: 8.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:9 and a light chain having at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identity to SEQ ID NO:10.

[0049] [Table 1-1]

[0050] [Table 1-2]

[0051] [Table 1-3]

[0052] [Table 1-4]

[0053] [Table 1-5]

[0054] [Table 1-6]

[0055] In some embodiments, the method described herein comprises identifying an antibody or fragment thereof for use in treating a mental or central nervous system disease or disorder. The antibody or fragment thereof can be identified by assaying for affinity to an antigen peptide of a pathogenic organism. The antibody or fragment thereof can be identified by assaying for affinity to the epitope LQNRKLV (SEQ ID NO: 41) of the NR1 subunit of the NMDA receptor.

[0056] In some embodiments, the pathogenic organism may include Toxoplasma, Paramecium, Campylobacter, Enterococcus, Peptoniphilus, Paenalcaligenes, Pseudomonas, Burkholderia, Chromobacterium, Acinetobacter, Paenibacillus, Escherichia, or Nocardia. In some embodiments, the pathogenic organism may be any one or more of the pathogenic organisms listed in Table 3.

[0057] In some embodiments, the method described herein includes identifying a subject as having been exposed to a pathogenic organism. The identifying step may include assaying a biological sample obtained from the subject for antibodies that bind to an immunogenic epitope of the pathogenic organism. The method may include identifying the subject as having one or more symptoms of a mental or central nervous system disease or disorder. The method may further include outputting a report identifying the subject as having an increased risk of N-methyl-D-aspartate (NMDA) receptor dysfunction.

[0058] In some embodiments, the one or more symptoms of a psychiatric or central nervous system disease or disorder may include delusions, seizures, speech disorders, movement difficulties, or aggression.

[0059] In some embodiments, the biological sample may be a fluid from a subject. The biological sample may include blood, sweat, saliva, cerebrospinal fluid (CSF), amniotic fluid, or mucus. In some embodiments, the biological sample includes blood, plasma, or serum. In some embodiments, the biological sample includes plasma. In some embodiments, the biological sample includes serum.

[0060] In some embodiments, the methods described herein may include identifying a subject as having a risk for an autoantibody-associated disease, comprising obtaining a biological sample from the subject, assaying the biological sample for antibodies that bind to the amino acid sequence LQNRKLV (SEQ ID NO: 41), and outputting a report identifying the subject as having a high or low risk for the autoantibody-associated disease.

[0061] In some embodiments, the methods disclosed herein include administering to a subject in need thereof an antibody or antigen-binding fragment thereof that binds to the subject's N-methyl-D-aspartate (NMDA) receptor.

[0062] In some embodiments, the subject is pregnant. In some embodiments, the risk of autoantibody-associated disease may be associated with the fetus of the pregnant subject. In some embodiments, the method comprises treating the pregnant subject for autoantibody-associated disease. In some embodiments, the treating comprises administering an FcRn receptor blocking compound to the subject.

[0063] In some embodiments, the FcRn receptor blocking compound can be a polypeptide. In some embodiments, the FcRn receptor blocking compound can be an antibody or a fragment thereof. In some embodiments, the FcRn receptor blocking compound can be selected from the group consisting of rozanolixizumab, SYNT001, M281, Argx-113, HL161-11G, HL161-11H, HL161-1A, DX-2504, DX-2507, ABY039, IMVT-1401 / RVT1401, and combinations thereof.

[0064] For example, to improve antibody affinity, modifications (e.g., substitutions) can be made in CDRs. Such modifications can be made in CDRs that encode codons with high mutation rates during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting variants can be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, for example, Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001)). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, for example, Cunningham and Wells Science, 244:1081-1085 (1989)). CDR-H3 and CDR-L3 are often targeted in particular. Alternatively, or in addition, the crystal structure of the antigen-antibody complex is targeted to identify the contact point between antibody and antigen.These contact residues and adjacent residues can be targeted or excluded as candidates for substitution.Variant can be screened to determine whether they have desired properties.

[0065] Amino acid sequence insertions and deletions include amino- and / or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme or polypeptide that extends the serum half-life of the antibody (e.g., for ADEPT). An example of an intrasequence insertional variant of an antibody molecule is an insertion of three amino acids in the light chain. An example of a terminal deletion is an antibody with a deletion of seven or fewer amino acids at the end of the light chain.

[0066] In some embodiments, an antibody is modified to increase or decrease its glycosylation (e.g., by modifying the amino acid sequence to create or remove one or more glycosylation sites). The carbohydrate attached to the Fc region of the antibody may also be modified. Natural antibodies from mammalian cells typically have Asn-binding domains in the CH2 domain of the Fc region. 297 (See, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharides can be various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, or fucose attached to GlcNAc in the stem of the biantennary oligosaccharide structure. Modification of oligosaccharides within antibodies can, for example, produce antibody variants with improved specific properties. Antibody glycosylation variants can have improved ADCC and / or CDC functions. In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose can be determined by the ratio of Asn to ATP. 297 Asn compared to the sum of all glycostructures attached to 297Asn is determined by calculating the average amount of fucose in the glycan (see, for example, WO 08 / 077546). 297 refers to the asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues, see e.g., Edelman et al. Proc Natl Acad Sci USA. 1969 May, 63(1):78-85). However, Asn 297 Also, due to slight sequence variations in antibodies, the fucosylated variants may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function (see, e.g., Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004) and Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)). Cell lines, e.g., knockout cell lines, and methods for their use can be used to produce defucosylated antibodies, e.g., Lec13 CHO cells and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells, which are deficient in protein fucosylation (see, e.g., Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)). Other antibody glycosylation variants are also included (see, e.g., U.S. Patent No. 6,602,684).

[0067] In some embodiments, the antibodies provided herein have a potency of about 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 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M to 10 -13 Dissociation constant (KD ) The antibody target may be an epitope target. D can be measured by any suitable assay. In certain embodiments, K D can be measured using a surface plasmon resonance assay (eg, using a BIACORE®-2000, a BIACORE®-3000, or an Octet).

[0068] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region, as used herein, refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. Fc regions include native-sequence Fc regions and variant Fc regions. An Fc region variant may comprise a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0069] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region herein refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. Fc regions include native-sequence Fc regions and variant Fc regions. An Fc region variant may comprise a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0070] In some examples, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP), which result in target cell killing, albeit through different mechanisms. Thus, in some embodiments, the antibodies described herein comprise a variable domain of the invention combined with a constant domain comprising a different Fc region selected based on the biological activity of the antibody for its intended use. In certain examples, human IgG can be classified into, for example, four subclasses, IgG1, IgG2, IgG3, and IgG4, each of which comprises an Fc region with a unique profile for binding to one or more of the Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32), FcγRIIIA and FcγRIIIB (CD16), and the inhibitory receptor FcγRIIB) and the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors, whereas IgG2 binds to FcγRIIA H131 binds to FcγRIIA R131 FcγRIIIA V158 IgG4 has low affinity for FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA. V158 The inhibitory receptor FcγRIIB binds to IgG1, IgG2, and IgG3 with lower affinity than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to either IgG2 or IgG4. As mentioned above, in general, with regard to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2.

[0071] In some embodiments, antibodies of the present disclosure are variants with reduced effector function, making them desirable candidates for applications in which certain effector functions (such as complement fixation and ADCC) are unnecessary or deleterious. Such antibodies may have reduced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, antibodies of the present disclosure are variants with increased effector function for applications in which increased immunogenicity is beneficial. Such antibodies may have increased CDC, ADCC, or ADCP, or a combination thereof. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays) may be used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and natural killer (NK) cells.

[0072] The antibody may have an extended half-life and improved binding to the fetal Fc receptor (FcRn) (see, e.g., U.S. Publication No. 2005 / 0014934). Such an antibody may comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn, and may comprise an Fc region with substitutions at one or more of the following Fc region residues according to the EU numbering system: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260 and 5,624,821, and WO 94 / 29351).

[0073] In some embodiments, it may be desirable to generate cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with a cysteine residue. In some embodiments, the substituted residues occur at accessible sites on the antibody. By positioning a reactive thiol group at a site for conjugation to another moiety, such as a drug moiety or a drug moiety of a linker, immunoconjugates can be generated. In some embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region.

[0074] In some embodiments, the antibodies provided herein can be further modified to include additional known and available nonproteinaceous moieties. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. Polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules.

[0075] The present disclosure also provides immunoconjugates comprising the anti-NR1 subunit antibodies described herein. An immunoconjugate is an antibody conjugated to one or more heterologous molecules. For example, the immunoconjugate may comprise an anti-NR1 subunit antibody bound to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a protein domain, a toxin (e.g., a protein toxin, bacterial, fungal, plant, or animal-derived, or fragment thereof), or a radioisotope. In some embodiments, the immunoconjugate may comprise an anti-NR1 antibody or a fragment thereof (e.g., scFv).

[0076] The antibodies described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to introduce a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a gene integration vector, or "integration vector," that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of an operably linked gene is referred to herein 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 used to regulate transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host is usually conferred by an origin of replication, and a selection gene may also be incorporated to facilitate recognition of transformants. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, and adeno-associated virus may also be used. Plasmid vectors can be linearized for integration into genomic regions. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, adenovirus, or adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus.

[0077] As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, can be determined using the formula 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). Such a formula has been incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence homology can be determined using the most recent version of BLAST as of the filing date of this application.

[0078] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transfect, or otherwise render suitable cells transgenic for the nucleic acid, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct, 2(5):466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing antibodies that is a Chinese hamster ovary cell (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of the cell useful for producing the antibody. In certain embodiments, described herein are methods of making an antibody, comprising culturing cells containing nucleic acid encoding the antibody under in vitro conditions sufficient to allow the production and secretion of the antibody.

[0079] In certain embodiments, described herein is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody having (i) a heavy chain amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and (ii) a light chain amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, or 10 integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the CHO cell line comprises a nucleic acid having any one of SEQ ID NOs: 42-51 integrated at a genomic location. In certain embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing with liquid nitrogen.

[0080] Also described herein are methods for producing the antibodies described herein. Such methods include incubating cells or cell lines containing nucleic acids encoding the antibodies in a cell culture medium under conditions sufficient to allow expression and secretion of the antibodies, and recovering the antibodies from the cell culture medium. The recovery step may further include one or more purification steps to remove viable cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain 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.

[0081] Treat, treatment, or treating, as used herein, refers to, for example, intentionally intervening in a physiological condition, resulting in a reduction in the severity of a disease or condition, shortening the duration of the disease course, alleviating or eliminating one or more symptoms associated with a disease or condition, or providing a beneficial effect to a subject with a disease or condition. Treatment does not require a cure of the underlying disease or condition.

[0082] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing the disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote regression of the disease can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0083] As used herein, "pharmaceutically acceptable" with respect to a "carrier," "excipient," or "diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0084] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Such salts include acid addition salts and base addition salts. 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 those derived from non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic sulfonic acids, and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0085] Treatment method In certain embodiments, antibodies useful for treating mental or central nervous system disorders or diseases are disclosed herein. Treatment refers to a method that seeks to improve or alleviate the condition being treated. With respect to mental or central nervous system disorders or diseases, treatment includes, but is not limited to, reducing one or more mental or central nervous system symptoms. In certain embodiments, treatment affects the severity of a mental or central nervous system disease or disorder. In certain embodiments, treatment encompasses use as a prophylactic or maintenance dose intended to prevent the recurrence or progression of a previously treated mental or central nervous system disease or disorder. It will be understood by those skilled in the art that not all individuals will respond equally or at all to an administered treatment, and that these individuals will nonetheless be considered to be treated.

[0086] In some embodiments, symptoms of a psychiatric or central nervous system disease or disorder may include seizures, delusions, speech disorders, movement difficulties, or aggression.

[0087] In certain embodiments, the disease or disorder of mental or central nervous system can include anti-NMDAR encephalitis, schizophrenia, psychosis, bipolar disorder, depression, epilepsy or dementia.In certain embodiments, the disease or disorder of mental or central nervous system is N-methyl-D-aspartate (NMDA) receptor dysfunction.In certain embodiments, the disease or disorder of mental or central nervous system that is treated recurs.

[0088] In certain embodiments, the method described herein comprises determining whether an individual has a pathogenic organism-reactive antibody in a biological sample from the individual, and then treating the individual against pathogen-associated autoantibodies with an antibody or antigen-binding fragment thereof that binds to the NMDAR1 (NR1) subunit of the NMDA receptor.In certain embodiments, exemplary pathogenic organisms are listed in Table 3.

[0089] In certain embodiments, the individual exhibits one or more symptoms associated with schizophrenia. In certain embodiments, the symptoms associated with schizophrenia include one or more of delusions, hallucinations, disorganized speech, or abnormal motor behavior. In certain embodiments, the individual has been diagnosed with schizophrenia. In certain embodiments, the individual is diagnosed with schizophrenia based on the Brief Cognitive Assessment Scale for Schizophrenia (BCASS) or the Positive and Negative Symptoms Scale for Schizophrenia (PANSS).

[0090] In some embodiments, pre-treatment and / or post-treatment clinical improvement is assessed using the BCASS, with the improvement in the BCASS score obtained after treatment. In some embodiments, after treatment, the BCASS is reduced by 90% or more. In some embodiments, after treatment, the BCASS is reduced by 80% or more. In some embodiments, after treatment, the BCASS is reduced by 70% or more. In some embodiments, after treatment, the BCASS is reduced by 60% or more. In some embodiments, after treatment, the BCASS is reduced by 50% or more. In some embodiments, after treatment, the BCASS is reduced by 40% or more. In some embodiments, after treatment, the BCASS total score is reduced by 30% or more. In some embodiments, after treatment, the BCASS is reduced by 20% or more. In some embodiments, after treatment, the BCASS is reduced by 10% or more. In some embodiments, after treatment, the BCASS is reduced by 5% or more.

[0091] In some embodiments, pre-treatment and / or post-treatment clinical improvement is assessed using the PANSS, with improvement in PANSS scores obtained after treatment. In some embodiments, after treatment, the PANSS is reduced by 90% or more. In some embodiments, after treatment, the PANSS is reduced by 80% or more. In some embodiments, after treatment, the PANSS is reduced by 70% or more. In some embodiments, after treatment, the PANSS is reduced by 60% or more. In some embodiments, after treatment, the PANSS is reduced by 50% or more. In some embodiments, after treatment, the PANSS is reduced by 40% or more. In some embodiments, after treatment, the PANSS total score is reduced by 30% or more. In some embodiments, after treatment, the PANSS is reduced by 20% or more. In some embodiments, after treatment, the PANSS is reduced by 10% or more. In some embodiments, after treatment, the PANSS is reduced by 5% or more.

[0092] In certain embodiments, the individual exhibits one or more symptoms associated with bipolar disorder. In certain embodiments, the symptoms associated with bipolar disorder include one or more of increased energy, agitation, impulsive behavior, agitation, lack of energy, feelings of worthlessness, low self-esteem, or suicidal thoughts. In certain embodiments, the individual has been diagnosed with bipolar disorder. In certain embodiments, the individual is diagnosed with bipolar disorder based on the Bipolar Assessment and Score Scale (BCASS) or the Bipolar Spectrum Diagnostic Scale (PANSS).

[0093] In some embodiments, pre-treatment and / or post-treatment clinical improvement is assessed using the BDRS, with improvement in BDRS scores obtained after treatment. In some embodiments, after treatment, the BDRS is 50 or less. In some embodiments, after treatment, the BDRS is 40 or less. In some embodiments, after treatment, the BDRS is 30 or less. In some embodiments, after treatment, the BDRS is 20 or less. In some embodiments, after treatment, the BDRS is 10 or less. In some embodiments, after treatment, the BDRS is 5 or less. In some embodiments, after treatment, the BDRS is 0. In some embodiments, the score of an individual receiving treatment is 1 point, 10 points, 20 points, 30 points, 40 points, or 50 points lower compared to the pre-treatment assessment.

[0094] In some embodiments, pre-treatment and / or post-treatment clinical improvement is assessed using the BSDS, with the improvement in the BSDS score obtained after treatment. In some embodiments, after treatment, the BSDS is 20 or less. In some embodiments, after treatment, the BSDS is 15 or less. In some embodiments, after treatment, the BSDS is 10 or less. In some embodiments, after treatment, the BSDS is 5 or less. In some embodiments, after treatment, the BSDS is 0. In some embodiments, the score of the treated individual is 1 point, 5 points, 10 points, 15 points, 20 points, or 25 points lower compared to the pre-treatment assessment.

[0095] In certain embodiments, the antibody or antigen-binding fragment thereof binds to the NMDAR1 (NR1) subunit of the NMDA receptor, binding to one or more amino acid residues contained within the amino acid sequence LQNRKLV (SEQ ID NO: 41). In certain embodiments, the antibody or antigen-binding fragment thereof that binds to the NMDAR1 (NR1) subunit of the NMDA receptor is any one or more of those disclosed in Table 1 of the present disclosure.

[0096] In certain embodiments, the methods described herein are not intended for the treatment of autoimmune encephalitis.In certain embodiments, the methods described herein are not intended for the treatment of NMDAR-related autoimmune encephalitis.

[0097] In certain embodiments, the antibody may be administered to a subject in need thereof by any route suitable for administering a pharmaceutical composition comprising the antibody, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered according to an appropriate dosing regimen, such as weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In certain embodiments, the antibody is administered once every three weeks. The antibody may be administered in any therapeutically effective amount. In certain embodiments, therapeutically acceptable amounts are greater than about 50 mg / kg, greater than about 75 mg / kg, greater than about 100 mg / kg, greater than about 125 mg / kg, greater than about 150 mg / kg, greater than about 175 mg / kg, and greater than about 200 mg / kg. In certain embodiments, a therapeutically acceptable amount is between about 0.1 mg / kg and about 200 mg / kg. In certain embodiments, a therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, a therapeutically acceptable amount is between about 1 mg / kg and about 20 mg / kg. In certain embodiments, a therapeutically acceptable amount is between about 1 mg / kg and about 10 mg / kg. In certain embodiments, a therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. In certain embodiments, a therapeutically acceptable amount is between about 5 mg / kg and about 20 mg / kg. A therapeutically effective amount includes an amount sufficient to alleviate one or more symptoms associated with the disease or affliction being treated.

[0098] Pharmaceutically Acceptable Excipients, Carriers, and Diluents In certain embodiments, the anti-NR1 NMDA receptor subunit antibody of the present disclosure is included in a pharmaceutical composition containing one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents may be included to increase the shelf life, stability, or manageability of the antibody. Such compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibody of the present disclosure is administered suspended in a sterile solution. In certain embodiments, the solution contains about 0.9% NaCl. In certain embodiments, the solution contains about 5.0% dextrose. In certain embodiments, the solution comprises one or more of a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid, methionine; or a chelating agent, e.g., EDTA or EGTA.

[0099] In certain embodiments, antibodies of the present disclosure can be transported / stored, lyophilized, and reconstituted prior to administration. In certain embodiments, lyophilized antibody formulations include a bulking agent such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation can be housed in a vial constructed of glass or other suitable non-reactive material. When formulated, the antibody, whether reconstituted or not, can be buffered at a specific pH, generally below 7.0. In certain embodiments, the pH can 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.

[0100] Also described herein are 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 devices for administration.

[0101] In certain embodiments, described herein are methods for preparing a treatment for a mental or central nervous system disorder or disease, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein are methods for preparing a treatment for cancer for storage or transportation, comprising lyophilizing one or more antibodies of the present disclosure.

[0102] The foregoing descriptions of specific embodiments of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and of course, many modifications and variations are possible in light of the above teachings. The embodiments were chosen and described to best explain the principles of the present disclosure and its practical application so as to enable others skilled in the art to best utilize the present disclosure and various embodiments, with various modifications as suited to the particular uses contemplated. It is intended that the scope of the present invention be defined by the appended claims and their equivalents.

[0103] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0104] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0105] Example 1: The predicted epitope of the NMDA receptor is LQNRKLV X-ray crystallography (Figures 1 and 2) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) (Figures 3, 4, 5, and 6) were performed on the binding complexes of autoantibodies and therapeutic antibodies specific for the NMDA receptor NR1 subunit to identify the binding epitopes of the NR1 subunit. X-ray crystallography: We used the Fab' fragment of a pathogenic autoantibody cloned from an anti-NMDA receptor encephalitis patient (#102Ab) and the NMDA receptor NR1 amino-terminal domain (ATD). Crystal structure of the NMDA receptor NR1 ATD complexed with #102A at 3.5 Å resolution was obtained. HDX-MS: We used a therapeutic one-armed antibody (ASP5803) or #102Ab and the NMDA receptor NR1 ATD. Unbound and antibody-bound NR1 ATD were incubated in deuterated (DO) water to exchange any amide hydrogens with deuterium from exposed amino acids of the protein. The positions of these deuterium molecules on the protein sequence were determined using high-resolution mass spectrometry. The predicted epitopes, verified by both HDX-MS and X-ray crystallography, can be seen in Table 2.

[0106] [Table 2]

[0107] The identified epitope, "LQNRKLV," was then identified in the sequence of the entire internal kinesin motor domain of Toxoplasma gondii (Figure 7, SEQ ID NO: 42).

[0108] Sequence of the entire internal kinesin motor domain from Toxoplasma gondii (T. gondii):

[0109]

[0110] As seen in Figures 8A and 8B, the "LQNRKLV" peptide was found in the NMDA receptor NR1 and the internal kinesin motor domain of T. gondii.

[0111] As summarized in Table 3 , the “LQNRKLV” peptide was found in parasite- or bacteria-encoded proteins associated with human neuropsychiatric disorders.

[0112] [Table 3-1]

[0113] [Table 3-2]

[0114] [Table 3-3]

[0115] Example 2: Toxoplasma / bacterial protein immunization to induce anti-NMDA receptor antibodies in mice Mice (Balb / c) were immunized intradermally four times every other week with 25 μg of Toxoplasma gondii or parasite / bacterial protein (25 μg emulsion) that shares 100% amino acid sequence (LQNRKLV) with the NR1 subunit of the NMDA receptor shown in Table 3 using TiterMax Gold. Two weeks later, three 25 μg boost injections of antigen protein (25 μg solution) containing CpG-B and alum were administered every other week. Blood samples were obtained from the right ear before each immunization. Serum was stored at -20°C.

[0116] Immunoglobulins (IgG / IgA / IgM) in murine sera induced by Toxoplasma or parasite / bacterial proteins will be assessed by ELISA binding assays (for both the original Toxoplasma / bacterial antigen and the NR1 subunit of the NMDA receptor) and NMDA receptor internalization assays.

[0117] ELISA binding assay: To detect murine IgG / IgA / IgM antibodies specific for Toxoplasma / parasite / bacterial proteins and NMDAR-NR1, microtiter plates were coated with Toxoplasma / parasite / bacterial proteins or NMDAR-NR1 (100 ng / well). All sera were titrated in Tris-buffered saline. Signals were detected with secondary antibodies: horseradish peroxidase-conjugated rabbit anti-mouse IgG, IgA, or IgM, diluted 1:5,000.

[0118] NMDA receptor internalization assay: Expression of the NR1 receptor was induced by culturing NMDA receptor-expressing HEK293 cells overnight in Induction Medium (Life Technologies Corporation, Carlsbad, CA, USA) consisting of Neurobasal Medium containing 10% dialyzed FBS, 50 U / mL penicillin-streptomycin, 2.0 μg / mL tetracycline, and 0.2 mM memantine. Cells were cultured at 1.5 × 10 cells / well in a 96-well plate. 5The cells are harvested and plated at 15 μL per well. Immunized murine serum is diluted and added to a total volume of 50 μL per well. The cells are incubated for 20 hours at 37°C and 5% CO2. After incubation, the cells are dissociated and transferred to a FACS reading plate (Corning, NY, USA). After washing the cells with FACS buffer (2% FBS in PBS), the cells are incubated on ice for 15 minutes with human Fc receptor binding inhibitor (20 μg / mL, Invitrogen, Carlsbad, CA, USA) and Dead cell stain kit (3:10,000, Invitrogen, Carlsbad, CA, USA). The NMDA receptors on the cell surface were then stained with ART5803 (5.0 μg / mL) or anti-KLH IgG1 (5.0 μg / mL) and phycoerythrin (PE)-conjugated goat anti-human IgG (1:100, Jackson ImmunoResearch Inc., West Grove, PA, USA). PE signal detection was performed using FACS Verse (BD Biosciences, Franklin Lakes, NJ, USA). FlowJo (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze flow cytometry data.

[0119] Immunization of mice with Toxoplasma and bacterial proteins, including LQNRKLV, resulted in the production of immunoglobulins (IgG, IgA, and IgM) that bind to the same Toxoplasma / parasite / bacteria antigen proteins. Due to the molecular homology between these antigens and the NMDA receptor NR1, these antibodies generated in mice by Toxoplasma / parasite / bacteria proteins bind to the NMDA receptor NR1 subunit in ELISA assays, but the binding titers of the antibodies in the immunized mouse sera were lower than those of the original Toxoplasma / parasite / bacteria antigens. The immunized mouse sera induced NMDA receptor internalization in NMDA receptor expressing HEK293 cells.

[0120] Example 3: Characterization of Toxoplasma-reactive T cells in patients with anti-NMDA receptor encephalitis and other psychiatric / cognitive disorders by ELISpot Patients with psychiatric / cognitive disorders, including anti-NMDA receptor encephalitis (ANRE), schizophrenia, depression, and dementia, as well as general encephalitis, have been shown to have higher titers of T. gondii than healthy controls. In the subsequent section on the role of T cells in the manifestations of these diseases, we refer to these disorders as psychotic disorders. Without being bound by theory, the relationship between T. gondii antibodies and psychotic disorders may stem from the numerous peptide commonalities and molecular homology between T. gondii proteins and NMDA receptors. Due to this extensive overlap in peptide sequences, it has been proposed that antibodies generated against the T. gondii proteome during the adaptive host response to infection may cross-react with subunits containing the NMDA receptor, leading to NMDA receptor dysfunction and downstream associated psychosis. In addition to antigen recognition, helper T cell assistance is generally required for naive B cells to evolve into antibody-secreting plasma cells.

[0121] Enzyme-linked immunosorbent spot (ELISpot) assays were performed to detect the presence or absence of cytokine secretion by T lymphocytes. Peripheral blood mononuclear cells (PBMCs) from psychiatric patients were enriched through Ficoll separation and subjected to ELISpot assays in which the cells were cultured in the presence of NMDA receptor mimetic T. gondii peptides, in addition to relevant control peptides, to determine overall responses to T. gondii antigens. Briefly, a panel of mimetic peptides was designed to be tested in the ELISpot assay. These peptides focused on the homologous segment shared between T. gondii and the NMDA receptor NR1 amino-terminal domain subunit. Peptides were designed as both HLA I preferred ligands for testing CD8 T lymphocyte responses and HLA II preferred ligands for testing CD4 T lymphocyte responses. For HLA class I, peptides were designed with 8–10 residues in length, and for HLA class II, peptides were designed with 13–25 residues in length (Table 4). The peptides were then co-cultured with PBMCs from patients with ANRE. Interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) responses are observed.

[0122] If psychotic disorders are mediated by cross-reactivity of T. gondii antibodies directed against NMDA receptors, patients with ANRE also possess clones of reactive T cells directed against T. gondii. Therefore, when PBMCs from psychotic patients are cultured in the presence of these peptides, antigen-mediated T cell responses are observed as long as there are sufficient T. gondii-reactive T cells to meet the sensitivity requirements of the ELISpot assay. These responses result in a host of cytokines expressed by the T cells, including IFNg and TNFa, which are visualized in the ELISpot system.

[0123] [Table 4-1]

[0124] [Table 4-2]

[0125] Example 4: Autologous mixed lymphocyte reaction with pulsed T. gondii peptides The mixed lymphocyte reaction (MLR) is a robust reaction that measures cooperation between cells of the immune system. Specifically, this reaction involves co-culturing bone marrow-derived dendritic cells (DCs) with T cells to elicit antigen-dependent responses from T cells. Dendritic cells (DCs) are pulsed with peptides of interest, which dock onto HLA class II receptors on their surface for presentation to autologous T cells. T cells harboring T cell receptors (TCRs) specific for these HLA / peptide combos are then stimulated through their TCRs to expand and proliferate. To measure proliferation of target cell populations, ELISpot assays (as described above) or flow cytometry using fluorescently labeled HLA peptide pentamers are used. The advantage of this technique over ELISpot is that autologous MLR allows T cell expansion for downstream analysis when T cells targeting the antigen of interest are present at low concentrations.

[0126] PBMCs are enriched by Ficoll separation of whole blood from patients with ANRE and other mental / cognitive disorders. Once enriched, T cells are isolated using a human T cell enrichment kit and magnetically separated from the remaining PBMC population. The T cells are then placed in culture conditions that support their proliferation, such as RPMI supplemented with cytokines such as IL2 and / or IL7. The remaining PBMC fraction is added to tissue-culture-treated culture flasks, where monocytes adhere after 2 hours while the remaining cell population remains in suspension. After 2 hours, the remaining suspended cells are removed, and the monocytes are placed in culture for 7 days supplemented with the cytokines IL4 and GMC-SF to differentiate into DCs.

[0127] Once T cells have expanded and autologous DCs have been generated, the two cell fractions are subjected to an MLR in which DCs are pulsed with a panel of T. gondii peptides that share sequence homology with the NR1 subunit of the NMDA receptor. T cells are removed at various time points and analyzed by ELISpot as described herein, in which isolated T cells are stimulated with each peptide antigen from the MLR assay, while a fraction is cryopreserved for downstream analysis using flow cytometry.

[0128] If a T cell population reactive to the HLA / T. gondii peptide combination exists, it is expanded by antigen-presenting cells, in this case DCs displaying a compatible HLA / peptide combination. After the MLR is complete, a subsequent ELISpot assay is performed to assess sufficient stimulation of T cells with each peptide species. Furthermore, proliferation of the target T cell population is measured by generating fluorescent HLA-matched peptide pentamers and analyzing each cryopreserved expanded T cell by flow cytometry.

[0129] Example 5: Characterization of NMDA receptor-homologous pathogenic peptide T cell responses in patients with anti-NMDA receptor encephalitis and other CNS disorders There are several exogenous peptide sequences produced by pathogenic species that share sequence homology with human NMDA receptor subunits. Without being bound by theory, it is believed that when an individual is infected with these pathogenic species, the individual's immune system generates a functional response that develops high-affinity antibodies against these pathogenic species in order to rid the host of them. Due to the sequence homology between some of these protein species and NMDA receptor subunits, cross-reaction of these antibodies against NMDA receptors can cause anti-NMDA receptor encephalitis (ANRE) and other mental / cognitive disorders. As mentioned in Example 3, B cell-produced antibodies generally require the assistance of helper cells, generally T cells, to class switch into antibody-secreting plasma cells.

[0130] Using HLA class I and class II matched peptide sequences, ELISpot and autologous MLR assays as described herein are performed to characterize the presence of T cells reactive to exogenous pathogen-produced peptides homologous to the NMDA receptor in patients with ANRE and other psychiatric / cognitive disorders.

[0131] If psychosis / dementia in a particular patient population is due to cross-reactivity of antibodies originally developed by the host to target exogenous NMDA receptor-homologous peptide sequences, T cells reactive to the pathogenic species will be present. If T cells are present in high concentrations, simply adding peptide to PBMC mixtures from these patients will activate them and detect them in ELISpot assays. If T cells are present in low concentrations, they can be expanded in autologous mixed lymphocyte reactions and characterized in downstream ELISpot and flow cytometry assays using fluorescent HLA / peptide pentamers.

[0132] Example 6: Cross-reactivity of antibodies from patients with CNS disorders to T. gondii and other organisms To determine whether a psychiatric or central nervous system disease or disorder is caused by the production of autoantibodies against the NR1 subunit of the NMDA receptor through infection with the parasite T. gondii or other organisms that further share a common epitope sequence, the binding of anti-NMDA receptor autoantibodies from patients with CNS disorders to recombinant T. gondii proteins, natural T. gondii proteins, and T. gondii peptides, as well as other recombinant proteins, natural proteins, and peptides derived from the organisms in Table 3, is assessed by ELISA, Western blot, and / or Bio-Layer Interferometry, as described herein.

[0133] IgG, IgA, and / or IgM monoclonal anti-NMDA receptor autoantibodies from patients with CNS disorders (anti-NMDA receptor encephalitis, schizophrenia, psychosis, bipolar disorder, depression, epilepsy, dementia, and encephalitis) will be evaluated for binding to recombinant T. gondii internal kinesin motor domain protein and proteins from other organisms in Table 3, including pathogenic antibody epitopes discovered in the NMDA receptor NR1 subunit (LQNRKLV).

[0134] If the antibodies of patients with CNS disorders are reactive to the recombinant proteins of Table 3, then there will be cross-reactivity of autoantibodies of patients with CNS disorders to these proteins in the binding assays described herein.

[0135] Because recombinant proteins may not exactly mimic the native protein conformation, patient-derived monoclonal autoantibodies are assessed for binding to cell lysates of the organisms in Table 3 by ELISA or Western blot.

[0136] If the antibodies of patients with CNS disorders are reactive to native T. gondii proteins, there will be cross-reactivity of the antibodies of patients with CNS disorders to these proteins in the binding assays described herein.

[0137] Since antigen fragments are primarily utilized in initiating immune responses, autoantibodies are assessed for binding to peptides generated by flanking epitope sequences with the organisms in Table 3, encompassing amino acids of various lengths up to 24 amino acids (example using the T gondii peptides in Table 4).

[0138] If the antibodies of patients with CNS disorders are reactive to T. gondii peptides, there will be cross-reactivity of the antibodies of patients with CNS disorders to these proteins in the binding assays described herein.

[0139] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention.

[0140] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in text incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

Claims

1. A one-armed antibody or antigen-binding fragment thereof that binds to an N-methyl-D-aspartate (NMDA) receptor in an individual's cells, for use in the treatment of an autoantibody-related mental or central nervous system disorder or disability, wherein the autoantibody-related mental or central nervous system disorder or disability is not encephalitis, and the one-armed antibody or antigen-binding fragment that binds to the NMDA receptor comprises scFv, Fab, Fab', and the antibody is not internalized in the individual's cells.

2. The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region (a) Heavy chain CDR1 described in SEQ ID NO: 23, heavy chain CDR2 described in SEQ ID NO: 24, heavy chain CDR3 described in SEQ ID NO: 25, light chain CDR1 described in SEQ ID NO: 26, light chain CDR2 described in SEQ ID NO: 27, and / or light chain CDR3 described in SEQ ID NO: 28 (b) Heavy chain CDR1 described in SEQ ID NO: 11, heavy chain CDR2 described in SEQ ID NO: 12, heavy chain CDR3 described in SEQ ID NO: 13, light chain CDR1 described in SEQ ID NO: 14, light chain CDR2 described in SEQ ID NO: 15, and / or light chain CDR3 described in SEQ ID NO: 16 (c) Heavy chain CDR1 described in SEQ ID NO: 17, heavy chain CDR2 described in SEQ ID NO: 18, heavy chain CDR3 described in SEQ ID NO: 19, light chain CDR1 described in SEQ ID NO: 20, light chain CDR2 described in SEQ ID NO: 21, and / or light chain CDR3 described in SEQ ID NO: 22 (d) Heavy chain CDR1 described in SEQ ID NO: 29, heavy chain CDR2 described in SEQ ID NO: 30, heavy chain CDR3 described in SEQ ID NO: 31, light chain CDR1 described in SEQ ID NO: 32, light chain CDR2 described in SEQ ID NO: 33, and / or light chain CDR3 described in SEQ ID NO: 34, (e) Heavy chain CDR1 described in SEQ ID NO: 35, heavy chain CDR2 described in SEQ ID NO: 36, heavy chain CDR3 described in SEQ ID NO: 37, light chain CDR1 described in SEQ ID NO: 38, light chain CDR2 described in SEQ ID NO: 39, and / or light chain CDR3 described in SEQ ID NO: 40 The antibody or antigen-binding fragment according to claim 1, comprising:

3. The antibody or antigen-binding fragment according to claim 2, wherein the heavy chain variable region comprises the same amino acid sequence as any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the light chain variable region comprises the same amino acid sequence as any one of SEQ ID NOs: 2, 4, 6, 8, or 10.

4. The antibody or antigen-binding fragment according to claim 2, wherein the heavy chain variable region comprises the same amino acid sequence as SEQ ID NO: 1, and the light chain variable region comprises the same amino acid sequence as SEQ ID NO:

2.

5. The antibody or antigen-binding fragment according to claim 2, wherein the heavy chain variable region comprises the same amino acid sequence as SEQ ID NO: 3, and the light chain variable region comprises the same amino acid sequence as SEQ ID NO:

4.

6. The antibody or antigen-binding fragment according to claim 2, wherein the heavy chain variable region comprises the same amino acid sequence as sequence number 5, and the light chain variable region comprises the same amino acid sequence as sequence number 6.

7. The antibody or antigen-binding fragment according to claim 2, wherein the heavy chain variable region comprises the same amino acid sequence as sequence number 7, and the light chain variable region comprises the same amino acid sequence as sequence number 8.

8. The use according to claim 2, wherein the heavy chain variable region includes the same amino acid sequence as sequence number 9, and the light chain variable region includes the same amino acid sequence as sequence number 10.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the autoantibody-related mental or central nervous system disorder or disorder is schizophrenia, psychosis, bipolar disorder, depression, epilepsy, or dementia.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the autoantibody-related mental or central nervous system disorder or disorder is schizophrenia.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment binds to the NMDAR1 (NR1) subunit of the NMDA receptor.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment binds to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41).

13. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8 for use in a method for treating or improving a mental or central nervous system disorder or disorder related to an autoantibody, wherein the method comprises a step of identifying an individual as having been exposed to a pathogenic organism, and the identifying step comprises identifying an antibody that binds to an immunogenic epitope of the pathogenic organism.

14. The pharmaceutical composition according to claim 13, wherein the pathogenic organism comprises one or more of the organisms listed in Table 3.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8 for use in a method for identifying an individual as being at risk of autoantibody-related mental or central nervous system disease or disorder, wherein the method is a) A step of assaying a biological sample from the said individual for an antibody that binds to the amino acid sequence LQNRKLV (SEQ ID NO: 41), b) Optionally, output a report indicating that the risk of autoantibody-related mental or central nervous system disease or disorder is high or low, or identify the subject. A pharmaceutical composition containing the following:

16. The pharmaceutical composition according to claim 15, wherein the biological sample comprises blood, sweat, saliva, cerebrospinal fluid (CSF), amniotic fluid, or mucus.

17. The pharmaceutical composition according to claim 15, wherein the individual is pregnant.

18. The pharmaceutical composition according to claim 17, wherein the risk of a mental or central nervous system disorder related to the autoantibody is related to the fetus of the individual.

19. The pharmaceutical composition according to any one of claims 15 to 18, wherein the method further comprises the step of treating the individual with respect to a mental or central nervous system disorder or disorder related to the autoantibody.

20. A method for identifying an antibody or antigen-binding fragment thereof contained in a pharmaceutical composition for use in the treatment of autoantibody-related mental or central nervous system disorders or conditions, comprising the step of identifying the antibody or antigen-binding fragment thereof that is specifically bound to the epitope LQNRKLV (SEQ ID NO: 41) of the NR1 subunit of the NMDA receptor.

21. The method according to claim 20, wherein the antibody or its antigen-binding fragment inhibits the binding of patient-derived autoantibodies that bind to the NMDA receptor.

22. The method according to claim 20, wherein the antibody or its antigen-binding fragment binds to the NR1 epitope at a KD of less than 1 mmol (mM).

23. The method according to claim 20, wherein the antibody or its antigen-binding fragment is humanized.

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