Methods and systems for identifying or treating central nervous system diseases

Abstract

Systems and methods for identifying autoantibodies associated with a central nervous system or psychiatric disease or disorder. Also disclosed herein are methods of treating a central nervous system or psychiatric disease or disorder associated with an autoantibody.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 369,923, filed on July 29, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Autoimmunity is the result that the body produces T cells and / or antibodies that are specific for self-antigens. This immune response can be triggered by environmental exposure (including viruses, pathogenic organisms or tumor growth). The immune response can become chronic, resulting in a variety of conditions, including mental and central nervous system (CNS) disorders or diseases caused by the production of autoantibodies. It is well known that viral, bacterial and parasitic infections, especially toxoplasmosis, increase the risk of schizophrenia and related mental disorders. A potential mechanism of infection-induced CNS diseases is molecular mimicry, in which the proteins of pathogenic organisms have similar sequences to those expressed in the human brain and produce cross-reactive autoantibodies. It is necessary to identify autoantibodies, related antigenic peptides and the treatment of diseases related to autoantibodies. Summary of the invention

[0004] 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 thereof an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain and light chain variable regions comprise: (a) a heavy chain CDR1 as shown in SEQ ID NO: 11, a heavy chain CDR2 as shown in SEQ ID NO: 12, a heavy chain CDR3 as shown in SEQ ID NO: 13, a light chain CDR1 as shown in SEQ ID NO: 14, a light chain CDR2 as shown in SEQ ID NO: 15, and / or a light chain CDR3 as shown in SEQ ID NO: 16; (b) a heavy chain CDR1 as shown in SEQ ID NO: 17, a heavy chain CDR2 as shown in SEQ ID NO: 18, a heavy chain CDR3 as shown in SEQ ID NO: 19, a light chain CDR1 as shown in SEQ ID NO: 20, a light chain CDR2 as shown in SEQ ID NO: 21, and / or a light chain CDR3 as shown in SEQ ID NO: 22; (c) a heavy chain CDR1 as shown in SEQ ID NO: 1 NO:23, a heavy chain CDR1 as shown in SEQ ID NO:24, a heavy chain CDR2 as shown in SEQ ID NO:25, a light chain CDR1 as shown in SEQ ID NO:26, a light chain CDR2 as shown in SEQ ID NO:27 and / or a light chain CDR3 as shown in SEQ ID NO:28; (d) a heavy chain CDR1 as shown in SEQ ID NO:29, a heavy chain CDR2 as shown in SEQ ID NO:30, a heavy chain CDR3 as shown in SEQ ID NO:31, a light chain CDR1 as shown in SEQ ID NO:32, a light chain CDR2 as shown in SEQ ID NO:33 and / or a light chain CDR3 as shown in SEQ ID NO:34; or (e) a heavy chain CDR1 as shown in SEQ ID NO:35, a heavy chain CDR2 as shown in SEQ ID NO:36, a heavy chain CDR3 as shown in SEQ ID NO:37, a light chain CDR1 as shown in SEQ ID NO:38, a heavy chain CDR2 as shown in SEQ ID NO:39, a light chain CDR3 as shown in SEQ ID NO:40 NO:39 and / or the light chain CDR2 as shown in SEQ ID NO:40. The heavy chain variable region may comprise an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO:1, 3, 5, 7 or 9, and the light chain variable region may comprise an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: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.

[0005] The mental or central nervous system disease or condition can be a condition associated with an autoantibody. The mental or central nervous system disease or condition can be schizophrenia. The mental or central nervous system disease or condition can be psychosis, bipolar disorder, depression, epilepsy and dementia. In some embodiments, the mental or central nervous system disease or condition is not encephalitis.

[0006] The antibody or its antigen binding fragment can bind to an antigen expressed by neurons and / or glial cells of the object. The antibody or its antigen binding fragment can bind to the N-methyl-D-aspartate (NMDA) receptor of the object. The antibody or its antigen binding fragment can bind to the NMDAR1 (NR1) subunit of the NMDA receptor. The antibody or its antigen binding fragment can bind to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41). The antibody or its antigen binding fragment can bind to at least one amino acid of NR1 consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41).

[0007] Disclosed herein is a method for identifying an antibody or antigen-binding fragment thereof for treating a mental or central nervous system disease or condition, the method comprising identifying an antibody or antigen-binding fragment thereof having the following properties: specifically 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 the binding of an autoantibody that binds to the NMDA receptor. The antibody or antigen-binding fragment thereof can bind to the NMDA receptor with a K of less than 1 mM. D Binds to an epitope. The antibody or antigen-binding fragment thereof may include a human antibody. The antibody or antigen-binding fragment thereof may be humanized.

[0008] Disclosed herein is a method for treating or improving a mental or central nervous system disease or condition, comprising identifying an object as having been exposed to a pathogenic organism, wherein the identification includes identifying an antibody bound to an immunogenic epitope of the pathogenic organism. The method may further include identifying the object as having one or more symptoms of a mental or central nervous system disease or condition. The method may further include outputting a report identifying the object as being at a high risk of N- methyl-D-aspartate (NMDA) receptor dysfunction. The mental or central nervous system disease or condition may be anti-NMDAR encephalitis, schizophrenia, psychosis, bipolar disorder, depression, epilepsy or dementia. The mental or central nervous system disease or condition is not encephalitis. The pathogenic organism may be from 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 organisms listed in Table 3. The one or more symptoms may include delusions, epileptic seizures, speech disorders, difficulty in moving or aggressive behavior.

[0009] Disclosed herein is a method for identifying a subject as having a risk of an autoantibody-related disease, comprising: obtaining a biological sample from the subject; determining antibodies in the biological sample 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 risk 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 an autoantibody-related disease may be associated with a fetus of the subject. The method may further include treating the subject for the autoantibody-related disease. The treatment 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 can be selected from the group consisting of: Rozanolixizumab, SYNT001, M281, Argx-113, HL161-11G, HL161-11H, HL161-1 A, DX-2504, DX-2507, ABY039, IMVT-1401 / RVT1401, and combinations thereof.

[0010] Disclosed herein is a method for treating or improving a mental or central nervous system disease or condition, comprising administering to a subject in need thereof an antibody or fragment that binds to an N-methyl-D-aspartate (NMDA) receptor of the subject. The antibody or its antigen binding fragment can bind to the NMDAR1 (NR1) subunit of the NMDA receptor. The antibody or its antigen binding fragment can bind to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41). The antibody or its antigen binding fragment can bind to at least one amino acid of NR1 consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41).

[0011] Incorporation by reference

[0012] 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 DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments utilizing the principles of the invention, wherein:

[0014] Figure 1The binding of pathogenic autoantibody fragments as described herein to the NR1 subunit of the NMDA receptor is shown.

[0015] Figure 2 The residues of the antibody fragments as described herein that interact with the amino acid residues of the NR1 subunit of the NMDA receptor are illustrated.

[0016] Figure 3 Graph depicting hydrogen deuterium exchange analysis of the binding of antibodies as described herein to the N-terminus of the NR1 subunit of the NMDA receptor.

[0017] Figure 4 The predicted epitope structure of the NR1 subunit of the NMDA receptor is shown.

[0018] Figure 5 HDX mass spectrometry analysis of anti-NR1 subunit and therapeutic antibodies is shown.

[0019] Figure 6 HDX mass spectrometry analysis of anti-NR1 subunit and pathogenic autoantibodies is shown.

[0020] Figure 7 The full internal kinesin motor domain protein sequence of Toxoplasma gondii is shown.

[0021] Fig. 8A and 8B Surface probability maps and associated antigenic indices of core epitope peptide sequences of the NMDA receptor NR1 subunit (8A) compared to Toxoplasma gondii kinesin (8B) are shown. DETAILED DESCRIPTION

[0022] In the following description, certain specific details are set forth in order to provide a comprehensive understanding of the various embodiments. However, those skilled in the art will appreciate that the embodiments provided can be implemented without these details. Unless the context otherwise requires, throughout the specification and the subsequent claims, the words "include" and their variants (such as "comprising") should be interpreted in an open, inclusive sense, i.e., "including but not limited to". As used in this specification and the appended claims, the singular forms "one", "a kind of" and "the / said" include plural references unless the content clearly states otherwise. It should also be noted that the term "or" is generally used in its meaning including "and / or", unless the content clearly states otherwise. In addition, the titles provided herein are only for convenience and do not explain the scope or meaning of the claimed embodiments.

[0023] Although certain embodiments and examples are disclosed below, the subject matter of the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as modifications and equivalents thereof. Therefore, the scope of the appended claims is not limited to any specific embodiment described below. For example, in any method or process disclosed herein, the actions or operations of the method or process may be performed in any suitable order and are not necessarily limited to any particular disclosed order. Various operations may be described in sequence as multiple discrete operations in a manner that may help understand certain embodiments, but the order of description should not be interpreted as implying that these operations are order-dependent. Additionally, the structures, systems, and / or devices described herein may be embodied as integrated components or separate components.

[0024] For the purpose 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 a group of advantages taught herein without having to achieve other aspects or advantages taught or suggested herein.

[0025] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly stated, all numbers may be interpreted as beginning with the word "about" or "approximately", even if the term does not appear explicitly. As used herein, the term "about" refers to an amount that is close to 10% or less of the amount described. In the following detailed description, reference is made to the accompanying drawings, which constitute a part of this document. In the drawings, similar symbols generally represent similar components unless the context otherwise provides. The illustrative embodiments described in the detailed description, the drawings, and the claims are not intended 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 is easy to understand that the aspects of the present disclosure as generally described herein and illustrated in the drawings may be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein. When describing a value and / or position, the phrase "about" or "approximately" may be used to indicate that the value and / or position described is within the reasonable expected range of the value and / or position. For example, a numerical value may have a value of + / -0.1% of the value (or value range), + / -1% of the value (or value range), + / -2% of the value (or value range), + / -5% of the value (or value range), + / -10% of the value (or value range), etc. Unless the context indicates otherwise, any numerical value given herein should also be understood to include about or approximately the value. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range described herein is intended to include all subranges contained therein. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values ​​are also disclosed, as appropriately understood by those skilled in the art. For example, if the value "X" is disclosed, "less than or equal to X" and "greater than or equal to X" (e.g., where X is a numerical value) are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and that the data represents endpoints and starting points, as well as ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15 are considered disclosed. It should also be understood that every unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0026] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations thereof (such as "comprising") indicate that various components may be employed together in methods and articles (e.g., compositions and devices including apparatus and methods). For example, the term "comprising" should be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0027] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease for which the compositions and methods described are used to treat. 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.

[0028] The antibodies provided include monoclonal antibodies and antibody fragments. Antibodies include antibody conjugates and molecules comprising antibodies, such as chimeric molecules. Therefore, antibodies include but are not limited to full-length and natural antibodies, as well as fragments and portions thereof that retain their binding specificity, such as any specific binding portion thereof, including those with any number of 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 entities). Monoclonal antibodies are generally one of the compositions of substantially homologous antibodies; therefore, except for naturally occurring mutations that may be present in small amounts, any individual antibody contained in the monoclonal antibody composition is identical. The monoclonal antibody may include a human IgG1 constant region. The monoclonal antibody may include a human IgG4 constant region.

[0029] The term "antibody" is used in the broadest sense herein and includes monoclonal antibodies, and includes complete 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 antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term covers genetic engineering and / or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heterologous conjugate antibodies, multispecific (e.g., bispecific) antibodies, double antibodies, three antibodies and four antibodies, tandem double-scFv, tandem three-scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments. The term also covers complete 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.

[0030] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", and are known in the art to refer to discontinuous amino acid sequences within an antibody variable region that confer antigen specificity and / or binding affinity. Generally speaking, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally speaking, there are 4 FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and 4 FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those by Kabat et al., (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme); 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 Plückthun 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 modelling 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.

[0031] The boundaries of a given CDR or FR may differ depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes are numbered based on the most common antibody region sequence lengths, with insertions accounted for by inserting letters, such as "30a", while deletions occur in some antibodies. The two schemes place certain insertions and deletions ("indels") in different locations, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0032] 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 native antibodies (V H and V L ) generally have a similar structure, each domain comprising four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen binding specificity. In addition, V domains from antibodies that bind to a specific antigen may be used. H or V L domain to separate antibodies that bind to the antigen and to screen for complementary V L or V H Libraries of domains (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0033] The specific binding or binding of antibody molecules described herein refers to the binding mediated by one or more CDR portions of the antibody. Not all CDRs may be required for specific binding. Specific binding can be demonstrated by, for example, ELISA for a specific target or antigen, and its binding is significantly increased compared to an isotype control antibody.

[0034] As described herein, "epitope" refers to a binding determinant of an antibody or fragment described herein, which is minimally required for the specific binding of the antibody or its antigen-binding fragment to the target antigen. When the target antigen is a polypeptide, the epitope will be a continuous or discontinuous epitope. A continuous epitope is formed by one region of the target antigen, while a discontinuous epitope can be formed by two or more separate regions. For example, a discontinuous epitope can be formed when the target antigen takes a tertiary structure that binds two amino acid sequences together and forms a three-dimensional structure bound by an antibody. When the target antigen is a polypeptide, an epitope is typically a plurality of amino acids connected to a polypeptide chain. A continuous epitope can include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids. Although an epitope can include a continuous polymer of amino acids, not every amino acid of the polymer can contact the amino acid residues of the antibody. Such uncontacted amino acids will still constitute part of the epitope because they may be important for the structure and connection of the contacted amino acids. For example, a skilled person can determine whether any given antibody binds to an epitope of a reference antibody by performing a cross-blocking experiment with the reference antibody. In certain embodiments, antibodies that bind to the same epitope of 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.

[0035] The antibodies provided include antibody fragments. "Antibody fragment" refers to a molecule other than a complete antibody, which contains a portion of the complete antibody that binds to the antigen to which 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 a specific embodiment, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as scFv.

[0036] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments comprising non-naturally occurring arrangements, such as fragments having two or more antibody regions or chains connected by synthetic joints (e.g., polypeptide joints) and / or fragments not produced by enzymatic digestion of naturally occurring intact antibodies. In some aspects, antibody fragments are scFv.

[0037] "Humanized" antibodies are antibodies 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. Humanized antibodies may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically 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 replaced by corresponding residues from a non-human antibody (e.g., an antibody derived from CDR residues), e.g., to restore or improve antibody specificity or affinity.

[0038] The antibodies provided include human antibodies. "Human antibodies" refer to antibodies with amino acid sequences corresponding to antibodies produced by humans or human cells, or antibodies of non-human origin utilizing human antibody libraries or other human antibody coding sequences (including human antibody libraries). The term does not include humanized forms of non-human antibodies comprising non-human antigen binding regions, such as antibodies in which all or substantially all CDRs are non-human.

[0039] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or is present outside the chromosome or randomly integrated into the chromosome of the animal. In such transgenic animals, the endogenous immunoglobulin locus is typically inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, which contain antibody coding sequences derived from human libraries.

[0040] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including antibodies and antibody chains provided, and other peptides, such as linkers and binding peptides, may include amino acid residues, including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, a polypeptide may include modifications relative to a natural or native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of a host producing the protein or errors due to PCR amplification. In some embodiments, amino acid sequence variants of antibodies provided herein are contemplated. Variants are generally different from the polypeptides specifically disclosed herein in terms of 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-mentioned polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptides as described herein and / or using any of many known techniques. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. The 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, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution can be performed to reach the final construct, as long as the final construct has the desired properties, such as antigen binding.

[0041] The percentage of sequence identity (%) relative to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence after the sequences are aligned and a gap is introduced (if necessary) to achieve the maximum percentage of sequence identity (and any conservative substitution is not considered as part of the sequence identity). The comparison for determining the purpose of amino acid sequence identity percentage 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. Appropriate parameters for aligning sequences can be determined, including algorithms required for achieving maximum alignment over the entire length of the compared sequences. However, for purposes herein, amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office (USCopyright Office) (Washington DC, 20559) with user documentation, and its U.S. Copyright Registration Number is TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not changed.

[0042] Where ALIGN-2 is used for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a particular percentage of amino acid sequence identity with a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless expressly stated otherwise, all percentage amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0043] In some embodiments, the methods described herein include treating or ameliorating a psychiatric or central nervous system disease or condition. In some embodiments, a psychiatric or central nervous system disease or condition is an autoantibody-related condition. In some embodiments, a psychiatric or central nervous system disease or condition is a neurodegenerative or cognitive disease or condition. In some embodiments, a psychiatric or central nervous system disease or condition may be schizophrenia, psychosis, bipolar disorder, depression, epilepsy, or dementia.

[0044] In some embodiments, the methods described herein include administering an antibody or antigen-binding fragment thereof that binds to neurons or glial cells to a subject in need thereof. In some embodiments, the antibody or its antigen-binding fragment binds to a neuron or glial cell receptor. In some embodiments, the neuron or glial cell 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 its antigen-binding fragment binds to the NMDAR1 (NR1) subunit of the NMDA receptor. In some embodiments, the antibody or its antigen-binding fragment binds to at least one amino acid of an epitope of NR1 consisting of the amino acid sequence LQNRKLV (SEQ ID NO: 41). In some embodiments, the antibody or its antigen-binding fragment binds to at least two amino acids of an epitope of NR1 consisting of the sequence LQNRKLV (SEQ ID NO: 41).

[0045] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least three amino acids of an epitope of NR1 consisting of the sequence LQNRKLV (SEQ ID NO: 41).

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO: 1, 3, 5, 7 or 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that has a CDR region that is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO: 11, 12, 13, 17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36 or 37.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain that is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO: 2, 4, 6, 8 or 10. In some embodiments, the antibody or fragment thereof comprises a light chain that has a CDR region that is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO: 14, 15, 16, 20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39 or 40.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions comprise: a heavy chain CDR1 as shown in SEQ ID NO:11, a heavy chain CDR2 as shown in SEQ ID NO:12, a heavy chain CDR3 as shown in SEQ ID NO:13, a light chain CDR1 as shown in SEQ ID NO:14, a light chain CDR2 as shown in SEQ ID NO:15 and / or a light chain CDR3 as shown in SEQ ID NO:16.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions comprise: a heavy chain CDR1 as shown in SEQ ID NO:17, a heavy chain CDR2 as shown in SEQ ID NO:18, a heavy chain CDR3 as shown in SEQ ID NO:19, a light chain CDR1 as shown in SEQ ID NO:20, a light chain CDR2 as shown in SEQ ID NO:21 and / or a light chain CDR3 as shown in SEQ ID NO:22.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions comprise: a heavy chain CDR1 as shown in SEQ ID NO:23, a heavy chain CDR2 as shown in SEQ ID NO:24, a heavy chain CDR3 as shown in SEQ ID NO:25, a light chain CDR1 as shown in SEQ ID NO:26, a light chain CDR2 as shown in SEQ ID NO:27 and / or a light chain CDR3 as shown in SEQ ID NO:28.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions comprise: a heavy chain CDR1 as shown in SEQ ID NO:29, a heavy chain CDR2 as shown in SEQ ID NO:30, a heavy chain CDR3 as shown in SEQ ID NO:31, a light chain CDR1 as shown in SEQ ID NO:32, a light chain CDR2 as shown in SEQ ID NO:33 and / or a light chain CDR3 as shown in SEQ ID NO:34.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions comprise: a heavy chain CDR1 as shown in SEQ ID NO:35, a heavy chain CDR2 as shown in SEQ ID NO:36, a heavy chain CDR3 as shown in SEQ ID NO:37, a light chain CDR1 as shown in SEQ ID NO:38, a light chain CDR2 as shown in SEQ ID NO:39 and / or a light chain CDR3 as shown in SEQ ID NO:40.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO: 1, 3, 5, 7, or 9 and a light chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO: 2, 4, 6, 8, or 10.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:1 and a light chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:2.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:3 and a light chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:4.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:5 and a light chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:6.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:7 or 9 and a light chain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:8.

[0058] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:9 and a light chain that is at least 80%, 85%, 90%, 95%, or 98%, 99%, or 100% identical to SEQ ID NO:10.

[0059] Table 1: Anti-NR1 antibody sequences

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] In some embodiments, the methods described herein include identifying antibodies or fragments thereof for treating mental or central nervous system diseases or conditions. Antibodies or fragments thereof can be identified by determining affinity with antigenic peptides of pathogenic organisms. Antibodies or fragments thereof can be identified by determining affinity with the epitope LQNRKLV (SEQ ID NO: 41) of the NR1 subunit of the NMDA receptor.

[0067] In some embodiments, the pathogenic organisms may include Toxoplasma, Paramecium, Campylobacter, Enterococcus, Proteophilus, Alcaligenes, Pseudomonas, Burkholderia, Chromobacterium, Acinetobacter, Paenibacillus, Escherichia or Nocardia. In some embodiments, the pathogenic organisms may be any one or more of the organisms listed in Table 3.

[0068] In some embodiments, the methods described herein include identifying the subject as having been exposed to a pathogenic organism. Identification may include determining antibodies that bind to immunogenic epitopes of pathogenic organisms in a biological sample obtained from the subject. The method may include identifying the subject as having one or more symptoms of a mental or central nervous system disease or condition. The method may further include outputting a report identifying the subject as being at high risk of N-methyl-D-aspartate (NMDA) receptor dysfunction.

[0069] In some embodiments, one or more symptoms of a psychiatric or central nervous system disease or disorder may include delusions, seizures, speech disorders, movement difficulties, or aggressive behavior.

[0070] In some embodiments, the biological sample can be a fluid derived from a subject. The biological sample can 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.

[0071] In some embodiments, the methods described herein may include identifying a subject as being at risk for an autoantibody-associated disease, comprising: obtaining a biological sample from the subject; determining in the biological sample an antibody that binds to the amino acid sequence LQNRKLV (SEQ ID NO:41); and outputting a report identifying the subject as being at high risk or low risk for the autoantibody-associated disease.

[0072] In some embodiments, the methods disclosed herein comprise administering to a subject in need thereof an antibody or fragment that binds to an N-methyl-D-aspartate (NMDA) receptor of the subject.

[0073] In some embodiments, the subject is pregnant. In some embodiments, the risk of autoantibody-related diseases can be associated with the fetus of the pregnant subject. In some embodiments, the method includes treating the autoantibody-related disease of the pregnant subject. In some embodiments, treatment can include administering an FcRn receptor blocking compound to the subject.

[0074] 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 fragment thereof. In some embodiments, the FcRn receptor blocking compound can be selected from: lolixizumab, SYNT001, M281, Argx-113, HL161-11G, HL161-11H, HL161-1 A, DX-2504, DX-2507, ABY039, IMVT-1401 / RVT1401, and combinations thereof.

[0075] Changes (e.g., substitutions) can be made in CDR, for example, to improve antibody affinity. Such changes can be made in CDR encoding codons with high mutation rates during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and the binding affinity of the resulting variants can be tested. 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., 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 usually particularly targeted. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0076] Amino acid sequence insertions and deletions include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, and intrasequence insertions and deletions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertion variants of antibody molecules include fusing the N-terminus or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or increasing the serum half-life of the antibody. Examples of intrasequence insertion variants of antibody molecules include inserting 3 amino acids in the light chain. Examples of terminal deletions include antibodies with 7 or fewer amino acids missing at the end of the light chain.

[0077] In some embodiments, the antibody is altered to increase or decrease its glycosylation (e.g., by changing the amino acid sequence so that one or more glycosylation sites are created or removed). The carbohydrate attached to the Fc region of the antibody can be altered. Natural antibodies from mammalian cells typically contain Asn attached to the CH2 domain of the Fc region via an N-linkage. 297 Branched biantennary oligosaccharides (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide can be a variety of carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, fucose attached to the GlcNAc in the stem of the biantennary oligosaccharide structure. For example, the oligosaccharides in the antibody can be modified to produce antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, the antibody variant has a carbohydrate structure lacking fucose attached to the Fc region (directly or indirectly). 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 is determined by the relative amount of fucose attached to Asn 297 The sum of all sugar structures is calculated as Asn 297 The average amount of fucose in the sugar chain at the position of Asn is determined (see, for example, WO08 / 077546). 297 refers to the asparagine residue at about position 297 of the Fc region (EU numbering of Fc region residues; see, e.g., Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78–85). However, due to minor sequence variations in antibodies, Asn 297It may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation 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 of using the same can be used to produce defucosylated antibodies, such as Lec13 CHO cells deficient in protein fucosylation and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (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. Pat. No. 6,602,684).

[0078] In some embodiments, the antibodies provided herein have a specificity for the antibody target 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 lower, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 The dissociation constant (K D ). The antibody target may be an epitope target. D Can be measured by any suitable assay. In certain embodiments, surface plasmon resonance assays (e.g., using or Octet) to measure K D .

[0079] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby generating Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain comprising at least a portion of the constant region. The Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0080] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby generating Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain comprising at least a portion of the constant region. The Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0081] In some cases, 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 cell-mediated phagocytosis (ADCP), which result in the killing of target cells, albeit by different mechanisms. Therefore, in some embodiments, the antibodies described herein comprise a combination of variable domains of the present invention and constant domains comprising different Fc regions, which are selected for intended use based on the biological activity of the antibody. In some cases, for example, human IgG can be divided into four subclasses: IgG1, IgG2, IgG3, and IgG4, and each of these subclasses comprises an Fc region with a unique spectrum for binding to one or more Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16) and inhibitory receptor FcγRIIB) and for the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA H131 Binds to FcγRIIA R131 FcγRIIIA V158 Lower affinity; gG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 Binding; and the inhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Same as above. Generally, in terms of ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2.

[0082] In some embodiments, the antibodies of the present disclosure are variants with reduced effector functions, which make them ideal candidates for applications where certain effector functions (e.g., complement fixation and ADCC) are unnecessary or harmful. Such antibodies may have reduced complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), or antibody dependent cellular phagocytosis (ADCP). In some embodiments, the antibodies of the present disclosure are variants with increased effector functions, for applications where increasing immunogenicity will be 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 molecules of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI TM and CytoTox Non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0083] The antibody may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such antibodies may comprise an Fc region having one or more substitutions therein to improve binding of the Fc region to FcRn, and include antibodies having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 according to the EU numbering system (see, e.g., U.S. Pat. No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0084] In some embodiments, it may be desirable to create a cysteine ​​engineered antibody, e.g., a "thioMAb," in which one or more residues of an antibody are replaced with a cysteine ​​residue. In some embodiments, the substituted residues occur at accessible sites of the antibody. Reactive thiol groups can be located at sites for conjugation with other moieties, such as drug moieties or linker drug moieties, to create immunoconjugates. In some embodiments, any one or more of the following residues can be replaced with 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.

[0085] In some embodiments, the antibodies provided herein can be further modified to include known and available additional non-protein parts. Suitable parts for antibody derivatization include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. Polymer can be any molecular weight, and can be branched or non-branched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be identical or different molecules.

[0086] The present disclosure also provides immunoconjugates comprising anti-NR1 subunit antibodies as described herein. An immunoconjugate is an antibody conjugated to one or more heterologous molecules. For example, an immunoconjugate may include an anti-NR1 subunit antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, protein domains, toxins (e.g., protein toxins, enzyme active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioactive isotopes. In some embodiments, an immunoconjugate may include an anti-NR1 antibody or a fragment thereof (e.g., scFv).

[0087] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are polynucleotide types comprising two or more nucleotide bases. In certain embodiments, nucleic acids are components of vectors, which can be used to transfer polynucleotides encoding polypeptides into cells. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a genomic integration vector or "integration vector", which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, such as a nucleic acid capable of extrachromosomal replication. The vector capable of directing the expression of a gene operably connected thereto is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In expression vectors, regulatory elements (such as promoters, enhancers, polyadenylation signals) for controlling transcription can be derived from mammals, microorganisms, viruses, or insect genes. The ability to replicate in a host (usually conferred by a replication origin) and selection genes that promote transformant recognition can be additionally incorporated. Vectors derived from viruses (such as slow viruses, retroviruses, adenoviruses, adeno-associated viruses, etc.) can 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, an adenovirus, or an 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.

[0088] As used herein, the terms "homology", "homology" or "homology percentage" when used 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, as modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formulas are incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The most recent version of BLAST as of the filing date of this application can be used to determine the homology percentage of a sequence.

[0089] Nucleic acids encoding antibodies described herein can be used to infect, transfect, transform or otherwise transduce cells appropriately for the nucleic acids, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures 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 cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines that can be used to produce antibodies, and are Chinese hamster ovary cells (CHO cells), NS0 mouse myeloma cells, or Cells. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell that can be used to produce the antibody. In certain embodiments, a method of preparing an antibody is described herein, comprising culturing a cell comprising a nucleic acid encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0090] In certain embodiments, a master cell bank is described herein, comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic position; 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 integrated at a genomic position, the antibody having (i) a heavy chain amino acid sequence shown in any one of SEQ ID NO: 1, 3, 5, 7 or 9; and (ii) a light chain amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8 or 10; 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 NO: 42-51 integrated at a genomic position. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding liquid nitrogen freezing.

[0091] Also described herein is a method for manufacturing antibodies as described herein. Such methods include incubating cells or cell lines comprising nucleic acids encoding antibodies in a cell culture medium under conditions sufficient to allow antibody expression and secretion, and further harvesting the antibody from the cell culture medium. Harvesting may further include one or more purification steps to remove living cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and culture medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification and / or ion exchange chromatography.

[0092] As used herein, "treatment" refers to intentional intervention with a physiological disease state, such as to reduce the severity of the disease or condition; shorten the duration of the course of the disease; improve or eliminate one or more symptoms associated with the disease or condition; or provide a beneficial effect to a subject suffering from the disease or condition. Treatment does not require a cure for the underlying disease or condition.

[0093] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes regression of a disease, as manifested by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability caused by the disease. The ability of a therapeutic agent to promote regression of a disease can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays.

[0094] As used herein, with respect to a "carrier," "excipient," or "diluent," "pharmaceutically acceptable" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In certain 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.

[0095] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not produce any adverse toxicological effects (see, for example, Berge, SM et al., (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids (such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc.) and non-toxic organic acids (such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Base addition salts include salts derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, etc.) and non-toxic organic amines (such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0096] Treatment

[0097] In certain embodiments, disclosed herein are antibodies that can be used to treat mental or central nervous system disorders or diseases. Treatment refers to a method of seeking to improve or alleviate the treated disorder. For mental or central nervous system diseases or conditions, treatment includes but is not limited to alleviating one or more symptoms of mental or central nervous system disorders. In certain embodiments, treatment will affect the severity of mental or central nervous system diseases or disorders. In certain embodiments, treatment covers the prevention or maintenance dose used as a recurrence or progression of a mental or central nervous system disease or disorder intended to prevent a previously treated mental or central nervous system disease or disorder. It will be appreciated by those skilled in the art that not all individuals will have the same response to the administered treatment or will not respond at all, nevertheless, these individuals are considered to be treated.

[0098] In some embodiments, symptoms of a psychiatric or central nervous system disease or disorder may include seizures, delusions, speech disorders, movement difficulties, or aggressive behavior.

[0099] In certain embodiments, mental or central nervous system diseases or conditions include anti-NMDAR encephalitis, schizophrenia, psychosis, bipolar disorder, depression, epilepsy or dementia. In certain embodiments, mental or central nervous system diseases or conditions are N-methyl-D-aspartate (NMDA) receptor dysfunction. In certain embodiments, the mental or central nervous system diseases or conditions treated are recurrent.

[0100] In certain embodiments, the methods described herein include determining in a biological sample from an individual whether the individual has pathogenic organism reactive antibodies, and subsequently treating the individual with 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.

[0101] In certain embodiments, the individual exhibits one or more symptoms associated with schizophrenia. In certain embodiments, the symptom associated with schizophrenia includes one or more of delusion, hallucination, speech disorder or motor behavior abnormality. In certain embodiments, the individual has been diagnosed with schizophrenia. In certain embodiments, according to the Brief Clinical Assessment Scale for Schizophrenia (BCASS) or the Positive and Negative Syndrome Scale for Schizophrenia (PANSS) for schizophrenia, the individual is diagnosed with schizophrenia.

[0102] In some embodiments, the clinical improvement before and / or after treatment is assessed using BCASS, wherein an improvement in the BCASS score is obtained after treatment. In some embodiments, after treatment, BCASS is reduced by 90% or more. In some embodiments, after treatment, BCASS is reduced by 80% or more. In some embodiments, after treatment, BCASS is reduced by 70% or more. In some embodiments, after treatment, BCASS is reduced by 60% or more. In some embodiments, after treatment, BCASS is reduced by 50% or more. In some embodiments, after treatment, 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, BCASS is reduced by 20% or more. In some embodiments, after treatment, BCASS is reduced by 10% or more. In some embodiments, after treatment, BCASS is reduced by 5% or more.

[0103] In some embodiments, PANSS is used to assess clinical improvement before and / or after treatment, wherein an improvement in PANSS score is obtained after treatment. In some embodiments, after treatment, PANSS is reduced by 90% or more. In some embodiments, after treatment, PANSS is reduced by 80% or more. In some embodiments, after treatment, PANSS is reduced by 70% or more. In some embodiments, after treatment, PANSS is reduced by 60% or more. In some embodiments, after treatment, PANSS is reduced by 50% or more. In some embodiments, after treatment, PANSS is reduced by 40% or more. In some embodiments, after treatment, PANSS total score is reduced by 30% or more. In some embodiments, after treatment, PANSS is reduced by 20% or more. In some embodiments, after treatment, PANSS is reduced by 10% or more. In some embodiments, after treatment, PANSS is reduced by 5% or more.

[0104] In certain embodiments, the individual exhibits one or more symptoms associated with bipolar disorder. In certain embodiments, the symptoms associated with bipolar disorder include increased energy, excitement, impulsive behavior, agitation, lack of energy, feeling worthless, low self-esteem or one or more of suicidal thoughts. In certain embodiments, the individual has been diagnosed with bipolar disorder. In certain embodiments, based on the Bipolar Depression Rating Scale (BDRS) or the Bipolar Spectrum Diagnostic Scale (BSDS), the individual is diagnosed as suffering from bipolar disorder.

[0105] In some embodiments, the clinical improvement before and / or after treatment is assessed using the BDRS, wherein an improvement in the BDRS score is 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 the treated individual will be reduced by 1, 10, 20, 30, 40, or 50 points compared to the pre-treatment assessment.

[0106] In some embodiments, the clinical improvement before and / or after treatment is assessed using the BSDS, wherein an improvement in the BSDS score is 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 will be reduced by 1, 5, 10, 15, 20, or 25 points compared to the pre-treatment assessment.

[0107] In certain embodiments, the antibody or antigen-binding fragment thereof that binds to the NMDAR1 (NR1) subunit of the NMDA receptor binds to one or more amino acid residues contained in 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.

[0108] In certain embodiments, the methods described herein are not used to treat autoimmune encephalitis. In certain embodiments, the methods described herein are not used to treat NMDAR-associated autoimmune encephalitis.

[0109] In certain embodiments, the antibody can be administered to a subject in need by any route suitable for administering a pharmaceutical composition containing 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 in a suitable dosage regimen, such as weekly, twice a week, monthly, twice a month, once every two weeks, once every three weeks, or once a month. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is greater than about 50 mg / kg, 75 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, and 200 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 200 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 20 mg / kg. The therapeutically effective amount includes an amount that is enough to improve one or more symptoms associated with the disease to be treated or the ailment.

[0110] Pharmaceutically acceptable excipients, carriers and diluents

[0111] In certain embodiments, the anti-NR1 NMDA receptor subunit antibodies of the present disclosure are contained in a pharmaceutical composition comprising 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 applicability of the antibody. Such compounds include salts, pH buffers, detergents, anticoagulants and preservatives. In certain embodiments, the antibodies of the present disclosure are suspended in a sterile solution for administration. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffer such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid such as glycine or arginine; an antioxidant such as ascorbic acid, methionine; or a chelating agent such as EDTA or EGTA.

[0112] In certain embodiments, the antibodies of the present disclosure can be transported / stored and reconstituted in a lyophilized form before administration. In certain embodiments, the lyophilized antibody formulation comprises a swelling agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40 or a combination thereof. The lyophilized formulation can be contained in a vial made of glass or other suitable non-reactive materials. The antibody, when formulated, can be buffered at a specific pH, usually less than 7.0, regardless of whether it is reconstituted. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0 or 5.0 and 6.0.

[0113] Also described herein are kits comprising, in a suitable container, one or more of the antibodies described herein and one or more additional components selected from the group consisting of: instructions for use; diluents, excipients, carriers, and devices for administration.

[0114] In certain embodiments, described herein is a method of preparing a treatment for a psychiatric 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 is a method of preparing a cancer treatment for storage or transport comprising lyophilizing one or more antibodies of the present disclosure.

[0115] The foregoing descriptions of specific embodiments of the present disclosure are presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. These embodiments are selected and described in order to best explain the principles of the invention and its practical application, thereby enabling those skilled in the art to best utilize the disclosure and consider various embodiments with various modifications suitable for a particular use. The scope of the present invention is intended to be defined by the appended claims and their equivalents.

[0116] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now appreciate that many variations, changes, and substitutions will not depart from the present invention. It should be understood that in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be employed. The following claims are intended to define the scope of the present invention and to cover methods and structures within the scope of these claims and their equivalents.

[0117] Example

[0118] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0119] Example 1: The predicted epitope of NMDA receptor is LQNRKLV

[0120] X-ray crystallography was performed on the binding complexes of autoantibodies and therapeutic antibodies specific for the NR1 subunit of the NMDA receptor. Figure 1 , Figure 2 ) and hydrogen deuterium exchange mass spectrometry (HDX-MS) ( Figure 3 , Figure 4 , Figure 5 , Figure 6 ) to identify the binding epitope of the NR1 subunit. X-ray crystallography: using pathogenic autoantibodies cloned from anti-NMDA receptor encephalitis patients (#102Ab) Fab' and NMDA receptor NR1 amino-terminal domain (ATD). Crystal structure of NMDA receptor NR1 ATD in complex with #102Ab at a resolution of 1.5 Å. HDX-MS: A therapeutic one-armed antibody (ASP5803) or #102Ab and NMDA receptor NR1 ATD were used. Unbound NR1 ATD and antibody-bound NR1 ATD were incubated in deuterated water (D2O) to exchange any amide hydrogens on exposed amino acids on the protein backbone with deuterium. High-resolution mass spectrometry was used to determine the location of these deuterated molecules on the protein sequence. Predicted epitopes validated by HDX-MS and X-ray crystallography can be found in Table 2.

[0121] Table 2: NR1 binding regions identified by HDX-MS and X-ray crystallography

[0122]

[0123] The identified epitope "LQNRKLV" was subsequently identified in the sequence of the full internal kinesin motor domain of Toxoplasma gondii ( Figure 7 , SEQ ID NO:42).

[0124] Sequence of the full internal kinesin motor domain of T. gondii:

[0125]

[0126] like Fig. 8A and Figure 8B It can be seen that the "LQNRKLV" peptide was found in the NMDA receptor NR1 and the internal kinesin motor domain of Toxoplasma gondii.

[0127] As summarized in Table 3, the "LQNRKLV" peptide was found in proteins encoded by parasites or bacteria associated with human neuropsychiatric disorders.

[0128] Table 3: Pathogenic species with homologous sequences to NMDA receptor subunits (only 100% epitope match with at least one publication supporting (at the genus level) any psychiatric, CNS or direct neurological significance)

[0129]

[0130]

[0131]

[0132] Example 2: Immunization of mice with Toxoplasma / bacterial proteins to induce anti-NMDA receptor antibodies

[0133] Mice (Balb / c) were immunized with Toxoplasma gondii or parasite / bacterial proteins (25ug emulsion) that share 100% amino acid sequence (LQNRKLV) with the NR1 subunit of the NMDA receptor as shown in Table 3 by intradermal injection four times every two weeks with TiterMax Gold. Two weeks later, booster immunizations were injected three times every two weeks with antigenic proteins (25ug solution) with CpG-B and alum. Blood samples were obtained from the right ear before each immunization. Serum was stored at -20°C.

[0134] Immunoglobulins (IgG / IgA / IgM) induced by T. gondii or parasite / bacterial proteins were assessed in mouse sera using ELISA binding assays (for native T. gondii / bacterial antigens and the NR1 subunit of the NMDA receptor) and NMDA receptor internalization assays.

[0135] ELISA binding assay: To detect mouse IgG / IgA / IgM antibodies specific for Toxoplasma / parasite / bacterial proteins and NMDAR-NR1, microtiter plates (100 ng / well) were coated with Toxoplasma / parasite / bacterial proteins or NMDAR-NR1. All sera were titrated with Tris-buffered saline. Secondary antibody horseradish peroxidase-labeled rabbit anti-mouse IgG, IgA or IgM was diluted 1:5,000 to detect the signal.

[0136] NMDA receptor internalization assay: HEK293 cells expressing NMDA receptors were cultured overnight in an induction medium consisting of Neurobasal medium (Life Technologies Corporation, Carlsbad, CA, USA) containing 10% dialyzed FBS, 50 U / mL penicillin-streptomycin, 2.0 μg / mL tetracycline, and 0.2 mM dimethylammonium to induce NR1 receptor expression. Cells were harvested and cultured at 1.5 x 10 5 Cells / 15 μL were plated in 96-well plates. The immune mouse serum was diluted and added in a total volume of 50 μL / well. The cells were incubated at 37°C, 5% CO2 for 20 hours. After incubation, the cells were dissociated and transferred to a FACS reading plate (Corning; NY, USA). After washing the cells with FACS buffer (2% FBS in PBS), the cells were incubated on ice for 15 minutes with human Fc receptor binding inhibitor (20 μg / mL; Invitrogen, Carlsbad, CA, USA) and a dead cell staining kit (3:10,000; Invitrogen, Carlsbad, CA, USA). Then, 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) were used to stain the NMDA receptors on the cell surface. PE signal detection was performed using FACS Verse (BD Biosciences, Franklin lakes, NJ, USA). FlowJo (BD Biosciences, Franklin lakes, NJ, USA) was used for analysis of flow cytometry data.

[0137] Immunization of mice with Toxoplasma and bacterial proteins containing LQNRKLV will result in the production of immunoglobulins (IgG / IgA / IgM) to bind to the same Toxoplasma / parasite / bacteria antigenic proteins. Due to molecular mimicry between these antigens and the NMDA receptor NR1, these antibodies generated in mice by Toxoplasma / parasite / bacteria proteins will bind to the NR1 subunit of the NMDA receptor in ELISA assays, although the antibody binding titers against the NR1 subunit of the NMDA receptor will be lower in the sera of immunized mice compared to the original Toxoplasma / parasite / bacteria antigens. Sera from immunized mice will induce NMDA receptor internalization in HEK293 cells expressing the NMDA receptor.

[0138] Example 3: Characterization of Toxoplasma gondii-reactive T cells in patients with anti-NMDA receptor encephalitis and other psychiatric / dementia conditions by ELISpot

[0139] Patients with anti-NMDA receptor encephalitis (ANRE), psychotic / dementia conditions (including schizophrenia, depression, dementia, and general encephalitis) have been shown to have higher titers against Toxoplasma gondii compared to healthy controls. In the subsequent chapters on the role of T cells in the manifestation of these conditions, these conditions are referred to as psychiatric disorders. Without being bound by theory, the connection between Toxoplasma gondii antibodies and psychiatric disorders can be derived from a large number of peptide commonalities and molecular mimicry between Toxoplasma gondii proteins and NMDA receptors. It has been suggested that due to this large amount of peptide sequence overlap, during the adaptive host response to infection, antibodies generated against the Toxoplasma gondii proteome may cross-react with subunits containing NMDA receptors, leading to NMDA receptor dysfunction, and downstream related psychosis. In addition to antigen recognition, naive B cells usually require helper T cell support in order to evolve and become plasma cells that secrete antibodies.

[0140] Enzyme-linked immunosorbent spot (ELISpot) assay was performed to detect the presence of cytokine secretion of T lymphocytes. Peripheral blood mononuclear cells (PBMC) obtained from patients with mental illness were separated and enriched by ficoll, and ELISpot assay was performed, wherein they were cultured in the presence of NMDA receptor mimicking Toxoplasma gondii peptides and related control peptides to determine their overall reactivity to Toxoplasma gondii antigens. In short, a group of mimetic peptides were designed to be tested in ELISpot assays. These peptides focus on the homologous fragments shared between Toxoplasma gondii and NMDA receptor NR1 amino terminal domain subunits. Peptides were designed as HLA I preferred ligands to test CD8 T lymphocyte reactivity, and HLA II preferred ligands were used to test CD4 T lymphocyte reactivity. For class I HLA, peptides were designed to have 8-10 residues, and for class II HLA, peptides were designed to have a length of 13-25 residues (Table 4). Peptides were co-cultured with PBMC of patients with ANRE. Interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) responses were observed.

[0141] If psychiatric disorders are mediated by cross-reactivity of T. gondii antibodies with the NMDA receptor, then patients with ANRE will additionally have reactive T cell clones against T. gondii. Therefore, when PBMCs from patients with psychosis are cultured in the presence of these peptides, antigen-mediated T cell responses will be observed as long as the number of T. gondii reactive T cells is sufficient to meet the sensitivity requirements of the ELISpot assay. These responses will result in the expression of a number of cytokines by the T cells, including IFNg and TNFa, which will be visualized in the ELISpot system.

[0142] Table 4: Sequence alignment of NMDA receptor NR1 with proposed Toxoplasma gondii peptide HLA ligands for (A) HLA class I and (B) HLA class II

[0143]

[0144]

[0145] Example 4: Autologous mixed lymphocyte reaction of pulsed Toxoplasma gondii peptide

[0146] The mixed lymphocyte reaction (MLR) is a robust reaction that measures cooperation between cells of the immune system. Specifically, it involves the co-culture of bone marrow-derived dendritic cells with T cells, with the goal of eliciting an antigen-dependent response from the T cells. Dendritic cells (DCs) are pulsed with peptides of interest, which dock onto class II HLA on their surface for presentation to autologous T cells. T cells containing TCRs specific for these HLA / peptide combinations are then stimulated through the T cell receptor (TCR) to expand and proliferate. To measure the 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 technology over ELISpot is that if the concentration of T cells targeting the antigen of interest is low, autologous MLR will achieve T cell expansion for downstream analysis.

[0147] Whole blood from patients with ANRE and other psychotic / dementia conditions will be subjected to ficoll separation to enrich PBMCs. Once PBMCs are enriched, T cells are isolated using a human T cell enrichment kit that separates T cells from the rest of the PBMC population by magnetism. T cells are then introduced into culture conditions that support their expansion, such as RPMI plus the addition of cytokines, such as IL2 and / or IL7. The remaining PBMC fractions are added to a culture bottle treated with tissue culture, and after two hours, monocytes adhere, while the remaining cell population remains in suspension. After two hours, the remaining suspended cells are removed, and the monocytes enter a 7-day culture supplemented with cytokines IL4 and GMC-SF to differentiate monocytes into DCs.

[0148] Once the T cells were expanded and autologous DCs were generated, two cell fractions were subjected to MLR in which the DCs were pulsed with a panel of T. gondii peptides with sequence homology to the NR1 subunit of the NMDA receptor. T cells were removed at different time points and analyzed by ELISpot as described herein, in which isolated T cells were stimulated with the respective peptide antigens from the MLR assay, while one fraction was cryopreserved for downstream analysis using flow cytometry.

[0149] If there is a population of T cells reactive to the HLA / T. gondii peptide combination, they will be expanded with the help of antigen presenting cells (in this case, DCs) displaying the compatible HLA / peptide combination. Once the MLR is complete, an ELISpot assay is then performed to assess whether their respective peptide species are sufficiently stimulating to the T cells. In addition, fluorescent HLA-matched peptide pentamers will be generated and the respective cryopreserved expanded T cells will be analyzed by flow cytometry to measure the expansion of the target T cell population.

[0150] Example 5: Characterization of NMDA receptor homologous pathogenic peptide T cell responses in patients with anti-NMDA receptor encephalitis and other CNS disorders

[0151] 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, its immune system will generate a functional response, in which high-affinity antibodies against these pathogenic species will be produced for the purpose of removing them from the host. Due to the sequence homology between several of these protein species and the subunits of the NMDA receptor, the cross-reactivity of these antibodies and the NMDA receptor may lead to anti-NMDA receptor encephalitis (ANRE) and other mental illnesses / dementia. As mentioned in Example 3, the B cells that produce antibodies usually need the assistance of helper cells (usually T cells) to be converted into plasma cells that secrete antibodies.

[0152] To characterize the presence of T cells reactive to exogenous, pathogenically produced peptides homologous to the NMDA receptor in patients with ANRE and other psychiatric / dementia conditions, ELISpot assays and autologous MLR assays as described herein were performed using class I and class II HLA compatible peptide sequences.

[0153] If psychosis / dementia in a particular patient population is due to cross-reactivity of antibodies originally generated by the host against target exogenous NMDA receptor homologous peptide sequences, then T cells reactive to the pathogenic species will be present. If the T cells are present at high concentrations, then simply adding the peptide to a mixture of PBMCs from these patients will result in T cell activation and will be detected in an ELISpot assay. If the T cells are present at low concentrations, then they will be expanded in an autologous mixed lymphocyte reaction and can be used for characterization in downstream ELISpot assays and flow cytometric assays using fluorescent HLA / peptide pentamers.

[0154] Example 6: Cross-reactivity of antibodies from patients with CNS disorders with Toxoplasma gondii and other organisms

[0155] To determine whether a psychiatric or central nervous system disease or disorder is caused by infection with the parasite Toxoplasma gondii or other organisms that additionally share common epitope sequences, resulting in the production of autoantibodies against the NMDA receptor NR1 subunit, the binding of anti-NMDA receptor autoantibodies in patients with CNS disorders to recombinant Toxoplasma gondii proteins, native Toxoplasma gondii proteins and Toxoplasma gondii peptides, as well as other recombinant proteins, native proteins and peptides derived from the organisms in Table 3, will be assessed by ELISA, Western blotting and / or biolayer interferometry as described herein.

[0156] IgG, IgA and / or IgM monoclonal anti-NMDA receptor autoantibodies derived from patients with CNS disorders (anti-NMDA receptor encephalitis, schizophrenia, psychosis, bipolar disorder, depression, epilepsy, dementia and encephalitis) were evaluated for binding to recombinant Toxoplasma gondii internal kinesin motor domain proteins and proteins from other organisms in Table 3 containing the pathogenic antibody epitope (LQNRKLV) found in the NMDA receptor NR1 subunit.

[0157] If CNS disorder patient antibodies are reactive to the recombinant proteins in Table 3, then CNS disorder patient autoantibodies will be cross-reactive to these proteins in the binding assays described herein.

[0158] Because recombinant proteins may not accurately mimic native protein conformation, binding of patient-derived monoclonal autoantibodies to cell lysates of the organisms in Table 3 was assessed by ELISA or Western blotting.

[0159] If the CNS disorder patient antibodies are reactive against native T. gondii proteins, then the CNS disorder patient antibodies will be cross-reactive against these proteins in the binding assays described herein.

[0160] Since antigen fragments are primarily used to initiate immune responses, binding of autoantibodies to peptides of varying lengths of amino acids (up to 24 amino acids) generated by flanking the epitope sequences of Table 3 organisms (e.g., the T. gondii peptides in Table 4) will be assessed.

[0161] If the CNS disorder patient antibodies are reactive to T. gondii peptides, then the CNS disorder patient antibodies will be cross-reactive to these proteins in the binding assays described herein.

[0162] Although this paper shows and describes this embodiment, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now expect many variations, changes and replacements without departing from the present invention. It should be understood that in practicing the present invention, various alternatives of the embodiments of the present invention described herein may be adopted.

[0163] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated herein by reference, 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 are inconsistent with definitions in this disclosure.

Claims

1. A method for treating or ameliorating a mental or central nervous system disease or condition, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain and light chain variable regions comprise: (a) a heavy chain CDR1 as set forth in SEQ ID NO: 11, a heavy chain CDR2 as set forth in SEQ ID NO: 12, a heavy chain CDR3 as set forth in SEQ ID NO: 13, a light chain CDR1 as set forth in SEQ ID NO: 14, a light chain CDR2 as set forth in SEQ ID NO: 15, and / or a light chain CDR3 as set forth in SEQ ID NO: 16; (b) a heavy chain CDR1 as set forth in SEQ ID NO: 17, a heavy chain CDR2 as set forth in SEQ ID NO: 18, a heavy chain CDR3 as set forth in SEQ ID NO: 19, a light chain CDR1 as set forth in SEQ ID NO: 20, a light chain CDR2 as set forth in SEQ ID NO: 21 and / or a light chain CDR3 as set forth in SEQ ID NO: 22; (c) a heavy chain CDR1 as set forth in SEQ ID NO:23, a heavy chain CDR2 as set forth in SEQ ID NO:24, a heavy chain CDR3 as set forth in SEQ ID NO:25, a light chain CDR1 as set forth in SEQ ID NO:26, a light chain CDR2 as set forth in SEQ ID NO:27, and / or a light chain CDR3 as set forth in SEQ ID NO:28; (d) a heavy chain CDR1 as set forth in SEQ ID NO:29, a heavy chain CDR2 as set forth in SEQ ID NO:30, a heavy chain CDR3 as set forth in SEQ ID NO:31, a light chain CDR1 as set forth in SEQ ID NO:32, a light chain CDR2 as set forth in SEQ ID NO:33 and / or a light chain CDR3 as set forth in SEQ ID NO:34; or (e) a heavy chain CDR1 as shown in SEQ ID NO:35, a heavy chain CDR2 as shown in SEQ ID NO:36, a heavy chain CDR3 as shown in SEQ ID NO:37, a light chain CDR1 as shown in SEQ ID NO:38, a light chain CDR2 as shown in SEQ ID NO:39 and / or a light chain CDR3 as shown in SEQ ID NO:

40.

2. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7 or 9, and the light chain variable region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8 or 10.

3. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7 or 9, and the light chain variable region comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8 or 10.

4. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7 or 9, and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8 or 10.

5. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7 or 9, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8 or 10.

6. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7 or 9, and the light chain variable region comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8 or 10.

7. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence identical to any one of SEQ ID NO: 1, 3, 5, 7 or 9, and the light chain variable region comprises an amino acid sequence identical to any one of SEQ ID NO: 2, 4, 6, 8 or 10.

8. The method according to any one of claims 1 to 7, wherein the psychiatric or central nervous system disease or disorder is an autoantibody-associated disorder.

9. The method according to any one of claims 1 to 7, wherein the psychiatric or central nervous system disease or disorder is schizophrenia.

10. The method of any one of claims 1 to 7, wherein the psychiatric or central nervous system disease or disorder is not encephalitis.

11. The method of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof binds to an antigen expressed by neurons of the subject.

12. The method of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof binds to an N-methyl-D-aspartate (NMDA) receptor of the subject.

13. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof binds to the NMDAR1 (NR1) subunit of the NMDA receptor.

14. The method of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof binds to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41).

15. The method of any one of claims 1 to 13, wherein 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).

16. A method for identifying an antibody or antigen-binding fragment thereof for treating a psychiatric or central nervous system disease or disorder, the method comprising identifying the antibody or antigen-binding fragment thereof that specifically binds to the epitope LQNRKLV (SEQ ID NO: 41) of the NR1 subunit of the NMDA receptor.

17. The method of claim 16, wherein the antibody or antigen-binding fragment thereof inhibits binding of an autoantibody that binds to the NMDA receptor.

18. The method of claim 16 or 17, wherein the antibody or antigen-binding fragment thereof has a K of less than 1 mM. D Binds to the epitope.

19. The method of any one of claims 16 to 18, wherein the antibody or antigen-binding fragment thereof comprises a human antibody.

20. The method of any one of claims 16 to 18, wherein the antibody or antigen-binding fragment thereof is humanized.

21. A method for treating or ameliorating a psychiatric or central nervous system disease or disorder comprising identifying a subject as having been exposed to a pathogenic organism, wherein said identifying comprises identifying an antibody that binds to an immunogenic epitope of said pathogenic organism.

22. The method of claim 21, further comprising identifying the subject as having one or more symptoms of a psychiatric or central nervous system disease or disorder.

23. The method of claim 21 or 22, further comprising outputting a report identifying the subject as being at high risk for N-methyl-D-aspartate (NMDA) receptor dysfunction.

24. The method of any one of claims 22 or 23, wherein the psychiatric or central nervous system disease or disorder is schizophrenia, psychosis, bipolar disorder, depression, epilepsy or dementia.

25. The method of any one of claims 22 or 23, wherein the psychiatric or central nervous system disease or disorder is not encephalitis.

26. The method of any one of claims 21 to 25, wherein the pathogenic organism comprises any one or more of the organisms listed in Table 3.

27. The method of any one of claims 21 to 25, wherein the one or more symptoms include delusions, seizures, speech disorders, movement difficulties, or aggressive behavior.

28. A method for identifying a subject as being at risk for an autoantibody-associated disease, comprising: a) obtaining a biological sample from the subject; b) determining antibodies in the biological sample that bind to the amino acid sequence LQNRKLV (SEQ ID NO: 41); and c) optionally, outputting a report or identifying the subject as being at high risk or low risk for the autoantibody-associated disease.

29. The method of claim 28, wherein the biological sample comprises blood, sweat, saliva, cerebrospinal fluid (CSF), amniotic fluid, or mucus.

30. The method of claim 28 or 29, wherein the subject is pregnant.

31. The method of any one of claims 28 to 30, wherein the risk of an autoantibody-associated disease is associated with a fetus of the subject.

32. The method of any one of claims 28 to 31, further comprising treating the subject for the autoantibody-associated disease.

33. The method of any one of claims 28 to 32, wherein the treatment comprises administering to the subject an FcRn receptor blocking compound.

34. The method of claim 33, wherein the FcRn receptor blocking compound is a polypeptide.

35. The method of claim 33, wherein the FcRn receptor blocking compound is an antibody or an antigen-binding fragment thereof.

36. The method of claim 35, wherein the FcRn receptor blocking compound is selected from the group consisting of: loliximab, SYNT001, M281, Argx-113, HL161-11 G, HL161-11H, HL161-1 A, DX-2504, DX-2507, ABY039, IMVT-1401 / RVT1401, and combinations thereof.

37. A method for treating or ameliorating a psychiatric or central nervous system disease or disorder, comprising administering to a subject in need thereof an antibody or fragment that binds to the subject's N-methyl-D-aspartate (NMDA) receptor.

38. The method of claim 37, wherein the antibody or antigen-binding fragment thereof binds to the NMDAR1 (NR1) subunit of the NMDA receptor.

39. The method of claim 37 or 38, wherein the antibody or antigen-binding fragment thereof binds to an epitope of NR1 comprising the amino acid sequence LQNRKLV (SEQ ID NO: 41).

40. The method of claim 37 or 38, wherein 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).

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