Anti-NMDA receptor antibody and application thereof

Abstract

The invention provides an anti-NMDA receptor antibody and application thereof. The anti-NMDA receptor antibody disclosed by the invention is specifically combined with a GluN1 subunit, a GluN2 subunit or a GluN3 subunit in an NMDA receptor, and is expected to be used for detecting the specificity of the anti-NMDA receptor antibody or detecting or diagnosing diseases related to autoimmune encephalitis.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically to an anti-NMDA receptor antibody and its applications. Background Technology

[0002] The N-methyl-D-aspartate receptor (NMDA receptor) is an ionotropic glutamate receptor composed of two GLUN1 subunits and two GLUN2 / 3 subunits. The NMDA receptor has eight splice variants of the GluN1 subtype and two splice variants of the GluN3 subunit (A, B) capable of binding to glycine; and four GluN2 subunits (A–D) capable of binding to glutamate. The GluN subunits contain two large extracellular domains: an N-terminal domain (ATD) and S1 and S2 domains, the S1 and S2 domains together forming the ligand-binding domain; three transmembrane domains (TM1, 3, 4); an intracellular polypeptide loop (TM2); and an intracellular C-terminal domain that connects to scaffold proteins and messenger systems. The NMDA receptor is crucial for learning and memory, while impaired receptor function is closely associated with conditions such as schizophrenia.

[0003] In 2005, Vitaliani, a foreign scholar, discovered an antigen in young female patients, primarily found in the cell membranes of hippocampal neurons, indicating a novel disease. These young women all had benign teratomas, and Vitaliani suggested this might be a new type of limbic paraneoplastic encephalitis. This encephalitis is an urgent condition with a potential for death, requiring long-term intensive care. However, most patients recovered and were discharged after tumor resection and immunotherapy. In 2007, Dalmau, another foreign scholar, also discovered anti-N-methyl-D-aspartate receptor (NMAD) antibodies in the cell membranes of hippocampal and prefrontal lobe neurons in patients with benign teratomas, naming it anti-NMDA receptor encephalitis. Anti-NMDA receptor encephalitis is an autoimmune syndrome caused by autoantibodies against NMDA receptors in the brain. In patients with anti-NMDA receptor encephalitis, IgGs recognize the extracellular N-terminal domain (ATD) of the GLUN1 subunit, and the ATD can regulate the function of NMDA receptor ion channels, including channel opening probability, inactivation rate, and allosteric regulation. Therefore, patients with this type of encephalitis may experience some mental and neurological symptoms, including memory loss, psychosis, hallucinations, epilepsy, and autonomic nervous system dysfunction. At the same time, some literature reports that a small number of IgGs from patients with anti-NMDA receptor encephalitis can recognize NR2A, NR2B, or NR2C.

[0004] Currently, the main clinical treatments for autoimmune encephalitis are plasma exchange and immunosuppression. However, the hybridoma antibody against GluN1 provided in this application can specifically block the binding of pathogenic antibodies to NMDA receptors, and has the potential to become a drug for treating anti-NMDA receptor autoimmune encephalitis. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-NMDA receptor antibody and its application.

[0006] In a first aspect of the invention, a heavy chain variable region of an antibody is provided, said heavy chain variable region comprising the following three complementarity-determining regions (CDRs):

[0007] HCDR1, as shown in SEQ ID NO:11,

[0008] HCDR2 shown in SEQ ID NO:12, and

[0009] HCDR3 as shown in SEQ ID NO:13; or

[0010] HCDR1, as shown in SEQ ID NO:17,

[0011] HCDR2 shown in SEQ ID NO:18, and

[0012] HCDR3 as shown in SEQ ID NO:19; or

[0013] HCDR1, as shown in SEQ ID NO:23,

[0014] HCDR2 shown in SEQ ID NO:24, and

[0015] HCDR3 as shown in SEQ ID NO:25; or

[0016] HCDR1, as shown in SEQ ID NO:29,

[0017] HCDR2 shown in SEQ ID NO:30, and

[0018] HCDR3 as shown in SEQ ID NO:31; or

[0019] HCDR1, as shown in SEQ ID NO:35,

[0020] HCDR2 shown in SEQ ID NO:36, and

[0021] HCDR3 as shown in SEQ ID NO:37; or

[0022] HCDR1, as shown in SEQ ID NO:43,

[0023] HCDR2 shown in SEQ ID NO:44, and

[0024] HCDR3 as shown in SEQ ID NO:45; or

[0025] HCDR1, as shown in SEQ ID NO:43,

[0026] HCDR2 shown in SEQ ID NO:44, and

[0027] HCDR3 as shown in SEQ ID NO:45;

[0028] Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

[0029] In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 41.

[0030] In a second aspect of the invention, a heavy chain of an antibody is provided, the heavy chain having the heavy chain variable region and heavy chain constant region described in the first aspect of the invention.

[0031] In another preferred embodiment, the heavy chain further includes a heavy chain constant region.

[0032] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0033] In another preferred embodiment, the heavy chain constant region is the human antibody heavy chain IgG1 constant region.

[0034] In a third aspect of the invention, a light chain variable region of an antibody is provided, the light chain variable region having a complementarity-determining region (CDR) selected from the group consisting of:

[0035] LCDR1 shown in SEQ ID NO:14

[0036] LCDR2 shown in SEQ ID NO:15, and

[0037] LCDR3 shown in SEQ ID NO:16; or

[0038] LCDR1 shown in SEQ ID NO:20

[0039] LCDR2 shown in SEQ ID NO:21, and

[0040] LCDR3 shown in SEQ ID NO:22; or

[0041] LCDR1 shown in SEQ ID NO:23

[0042] LCDR2 shown in SEQ ID NO:26, and

[0043] LCDR3 shown in SEQ ID NO:27; or

[0044] LCDR1 shown in SEQ ID NO:28

[0045] LCDR2 shown in SEQ ID NO:32, and

[0046] LCDR3 shown in SEQ ID NO:33; or

[0047] LCDR1 shown in SEQ ID NO:38

[0048] LCDR2 shown in SEQ ID NO:39, and

[0049] LCDR3 as shown in SEQ ID NO:40; or

[0050] LCDR1 shown in SEQ ID NO:46

[0051] LCDR2 shown in SEQ ID NO:47, and

[0052] LCDR3 as shown in SEQ ID NO:48;

[0053] Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

[0054] In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10 or 42.

[0055] In a fourth aspect of the invention, a light chain of an antibody is provided, said light chain having the light chain variable region and light chain constant region described in the third aspect of the invention.

[0056] In a fifth aspect of the invention, an antibody or an antigen-binding fragment thereof is provided, the antibody having:

[0057] (1) the heavy chain variable region as described in the first aspect of the invention; and / or

[0058] (2) The light chain variable region as described in the third aspect of the present invention;

[0059] Preferably, the antibody has: a heavy chain as described in the second aspect of the invention; and / or a light chain as described in the fourth aspect of the invention;

[0060] Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

[0061] In another preferred embodiment, the NMDA receptor includes a subunit selected from the group consisting of: GluN1 / GluN2A subunit, GluN1 / GluN2B subunit, GluN1 / GluN2C subunit, GluN1 / GluN2D subunit, GluN1 / GluN3A subunit, or GluN1 / GluN3B subunit.

[0062] In another preferred embodiment, the NMDA receptor is a tetramer.

[0063] In another preferred embodiment, the NMDA receptor comprises two GluN1 subunits and two GluN2 or GluN3 subunits; preferably, two GluN1 subunits and two GluN2A (or 2B, 2C or 2D) subunits; or two GluN1 subunits and two GluN3A (or 3B) subunits.

[0064] In another preferred embodiment, the antibody specifically binds to the GluN1 subunit of the NMDA receptor, i.e., "a specific antibody against the GluN1 subunit of the NMDA receptor".

[0065] In another preferred embodiment, the antibody specifically binds to subunits of the NMDA receptor other than the GluN1 subunit, i.e., "a specific antibody against the non-NMDA receptor GluN1 subunit".

[0066] In another preferred embodiment, the antibody specifically binds to the GluN2 or GluN3 subunit of the NMDA receptor.

[0067] In another preferred embodiment, the antibody is an antibody specifically targeting the GluN1 subunit, an antibody specifically targeting Glu2A, an antibody specifically targeting Glu2B, an antibody specifically targeting Glu2C, an antibody specifically targeting Glu2D, an antibody specifically targeting Glu3A, or an antibody specifically targeting Glu3B.

[0068] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (e.g., 1-3, more preferably 1-2, more preferably 1).

[0069] In another preferred embodiment, the derived sequence, which has been added, deleted, modified, and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity, is an amino acid sequence with homology or sequence identity of at least 96%.

[0070] In another preferred embodiment, the antibody further includes a heavy chain constant region and / or a light chain constant region.

[0071] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0072] In another preferred embodiment, the heavy chain constant region is the human antibody heavy chain IgG1 constant region, and the light chain constant region is the human antibody light chain kappa constant region.

[0073] In another preferred embodiment, the heavy chain variable region of the antibody further includes a human-derived frame region, and / or the light chain variable region of the antibody further includes a human-derived frame region.

[0074] In another preferred embodiment, the heavy chain variable region of the antibody further includes a mouse-derived frame region, and / or the light chain variable region of the antibody further includes a mouse-derived frame region.

[0075] In another preferred embodiment, the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

[0076] In another preferred embodiment, the antibody is a partially or fully humanized, or fully human, monoclonal antibody.

[0077] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0078] In another preferred embodiment, the antibody is a full-length antibody protein.

[0079] In another preferred embodiment, the antigen-binding fragment includes: (i) a Fab fragment; (ii) an F(ab')2 fragment; (iii) an Fd fragment; (iv) an Fv fragment; (v) a single-chain Fv (scFv) molecule; and (vi) a dAb fragment.

[0080] In another preferred embodiment, the antibody has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the following three complementarity-determining regions (CDRs):

[0081] HCDR1, as shown in SEQ ID NO:11,

[0082] HCDR2 shown in SEQ ID NO:12, and

[0083] HCDR3 as shown in SEQ ID NO:13; and

[0084] The light chain variable region includes the following three complementary determinant regions (CDRs):

[0085] LCDR1 shown in SEQ ID NO:14

[0086] LCDR2 shown in SEQ ID NO:15, and

[0087] LCDR3 shown in SEQ ID NO:16; or

[0088] The heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0089] HCDR1, as shown in SEQ ID NO:17,

[0090] HCDR2 shown in SEQ ID NO:18, and

[0091] HCDR3 as shown in SEQ ID NO:19; and

[0092] The light chain variable region includes the following three complementary determinant regions (CDRs):

[0093] LCDR1 shown in SEQ ID NO:20

[0094] LCDR2 shown in SEQ ID NO:21, and

[0095] LCDR3 shown in SEQ ID NO:22; or

[0096] The heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0097] HCDR1, as shown in SEQ ID NO:23,

[0098] HCDR2 shown in SEQ ID NO:24, and

[0099] HCDR3 as shown in SEQ ID NO:25; and

[0100] The light chain variable region includes the following three complementary determinant regions (CDRs):

[0101] LCDR1 shown in SEQ ID NO:26

[0102] LCDR2 shown in SEQ ID NO:27, and

[0103] LCDR3 shown in SEQ ID NO:28; or

[0104] The heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0105] HCDR1, as shown in SEQ ID NO:29,

[0106] HCDR2 shown in SEQ ID NO:30, and

[0107] HCDR3 as shown in SEQ ID NO:31; and

[0108] The light chain variable region includes the following three complementary determinant regions (CDRs):

[0109] LCDR1 shown in SEQ ID NO:32,

[0110] LCDR2 shown in SEQ ID NO:33, and

[0111] LCDR3 as shown in SEQ ID NO:34; or

[0112] The heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0113] HCDR1, as shown in SEQ ID NO:35,

[0114] HCDR2 shown in SEQ ID NO:36, and

[0115] HCDR3 as shown in SEQ ID NO:37; and

[0116] The light chain variable region includes the following three complementary determinant regions (CDRs):

[0117] LCDR1 shown in SEQ ID NO:38

[0118] LCDR2 shown in SEQ ID NO:39, and

[0119] LCDR3 as shown in SEQ ID NO:40; or

[0120] The heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0121] HCDR1, as shown in SEQ ID NO:43,

[0122] HCDR2 shown in SEQ ID NO:44, and

[0123] HCDR3 as shown in SEQ ID NO:45; and

[0124] The light chain variable region includes the following three complementary determinant regions (CDRs):

[0125] LCDR1 shown in SEQ ID NO:46

[0126] LCDR2 shown in SEQ ID NO:47, and

[0127] LCDR3 as shown in SEQ ID NO:48;

[0128] Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

[0129] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:2.

[0130] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4.

[0131] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:6.

[0132] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:8.

[0133] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:10.

[0134] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:42.

[0135] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, or 41 in the sequence listing.

[0136] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequences shown in SEQ ID NO:2, 4, 6, 8, 10, or 42 in the sequence listing.

[0137] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0138] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0139] In another preferred embodiment, the antibody has one or more properties selected from the group consisting of:

[0140] (a) Binding to NMDA receptors;

[0141] (b) Induces NMDA receptor endocytosis;

[0142] (c) Reduce the number of NMDA receptors on the synapse.

[0143] In another preferred embodiment, the antigen-binding fragment of the antibody has one or more characteristics selected from the group consisting of:

[0144] (a) Binding to NMDA receptors;

[0145] (b) It does not induce endocytosis of NMDA receptors;

[0146] (c) Competitive binding of anti-NMDA receptor antibodies to NMDA receptors.

[0147] In another preferred embodiment, the anti-NMDA receptor antibody is selected from the group consisting of:

[0148] Antibodies specifically targeting the GluN1 subunit, Glu2A, Glu2B, Glu2C, Glu2D, Glu3A, and Glu3B.

[0149] In another preferred embodiment, the antibody binds to the GluN1 subunit of the NMDA receptor; preferably, it binds to the NTD region of the GluN1 subunit.

[0150] In another preferred embodiment, the antibody binds to the GluN1 subunit of the NMDA receptor.

[0151] In another preferred embodiment, the antibody binds to the Glu2A subunit of the NMDA receptor; preferably, it binds to the LBD-S1 region of the Glu2A subunit.

[0152] In another preferred embodiment, the antibody binds to the Glu2B subunit of the NMDA receptor.

[0153] In another preferred embodiment, the antibody binds to the Glu2C subunit of the NMDA receptor; preferably, it binds to the NTD region of the Glu2C subunit.

[0154] In another preferred embodiment, the antibody binds to the Glu2D subunit of the NMDA receptor.

[0155] In another preferred embodiment, the antibody binds to the Glu3A subunit of the NMDA receptor.

[0156] In another preferred embodiment, the antibody binds to the Glu3B subunit of the NMDA receptor.

[0157] In another preferred embodiment, the binding is a specific binding.

[0158] In a sixth aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0159] (i) a sequence of the heavy chain variable region as described in the first aspect of the invention, a sequence of the heavy chain as described in the second aspect of the invention, a sequence of the light chain variable region as described in the third aspect of the invention, a sequence of the light chain as described in the fourth aspect of the invention, or a sequence of an antibody or an antigen-binding fragment thereof as described in the fifth aspect of the invention; and

[0160] (ii) Optional tag sequences to assist in expression and / or purification.

[0161] In another preferred embodiment, the tag sequence includes a 6His tag.

[0162] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0163] In another preferred embodiment, the recombinant protein is a monomer, dimer, trimer, tetramer, or polymer.

[0164] In another preferred embodiment, the recombinant protein comprises:

[0165] (i) An antibody selected from the group consisting of: the heavy chain variable region of the antibody containing the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region of the antibody containing the amino acid sequence shown in SEQ ID NO:2;

[0166] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4;

[0167] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:6;

[0168] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:8;

[0169] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:10;

[0170] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:42;

[0171] (ii) Optional tag sequences to assist in expression and / or purification.

[0172] In a seventh aspect of the invention, a polynucleotide is provided that encodes a polypeptide selected from the group consisting of:

[0173] (1) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody or antigen-binding fragment thereof as described in the fifth aspect of the present invention; or

[0174] (2) The recombinant protein as described in the sixth aspect of the present invention.

[0175] In an eighth aspect of the invention, a carrier is provided that contains the polynucleotides described in the seventh aspect of the invention.

[0176] In a ninth aspect of the invention, a genetically engineered host cell is provided, which contains a vector or genome according to the eighth aspect of the invention in which the polynucleotides according to the seventh aspect of the invention are integrated.

[0177] In a tenth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0178] (a) the heavy chain variable region as described in the first aspect of the invention, the heavy chain as described in the second aspect of the invention, the light chain variable region as described in the third aspect of the invention, the light chain as described in the fourth aspect of the invention, the antibody or antigen-binding fragment thereof as described in the fifth aspect of the invention, and the recombinant protein as described in the sixth aspect of the invention; and

[0179] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0180] In an eleventh aspect of the present invention, a method for detecting the specificity of anti-NMDA receptor antibodies in biological samples is provided, comprising:

[0181] (a) Provide a biological sample;

[0182] (b) Determine whether the test substance possesses properties selected from the group consisting of:

[0183] (i) It can bind to NMDA receptors;

[0184] (ii) It can interfere with or inhibit the binding of NMDA receptors to anti-NMDA receptor antibody standards; and

[0185] (c) For the test article that has the above-mentioned characteristics as tested in step (b), the test article is a competitive antibody against the anti-NMDA receptor antibody standard or an antibody that can inhibit or interfere with the binding between the anti-NMDA receptor antibody standard and the NMDA receptor.

[0186] In another preferred embodiment, the test substance is a potential therapeutic agent for NMDA receptor-related diseases.

[0187] In another preferred embodiment, the anti-NMDA receptor antibody standard is selected from the following group:

[0188] Antibodies specifically targeting the GluN1 subunit, Glu2A, Glu2B, Glu2C, Glu2D, Glu3A, and Glu3B.

[0189] In another preferred embodiment, the anti-NMDA receptor antibody is an antibody as described in the first aspect of the invention.

[0190] In another preferred embodiment, the NMDA receptor comprises two GluN1 subunits and two GluN2 or GluN3 subunits; preferably, two GluN1 subunits and two GluN2A (or 2B, 2C or 2D) subunits; or two GluN1 subunits and two GluN3A (or 3B) subunits.

[0191] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0192] Figure 1The left image shows the trajectory of GluN1 / Glu3A protein passing through a molecular sieve, and the right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining results.

[0193] Figure 2 The results showed that live-cell immunofluorescence staining was used to detect the subunit specificity of antibody 4F11 against GluN1.

[0194] Figure 3 The results show: A: FSEC detection results of anti-GluN1 antibody 4F11 and GluN1 / GluN2A, GluN1 / GluN2B, GluN1 / GluN2C, and GluN1 / GluN2D proteins; B: Cryo-electron microscopy three-dimensional reconstruction results of 4F11 Fab and GluN1 / GluN2A proteins.

[0195] Figure 4 The images show: A: Left image shows the trajectory of GluN1 / Glu2A protein through a molecular sieve, and right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining result; B: Left image shows the trajectory of GluN1 / Glu2B protein through a molecular sieve, and right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining result; C: Left image shows the trajectory of GluN1 / Glu2C protein through a molecular sieve, and right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining result; D: Left image shows the trajectory of GluN1 / Glu2D protein through a molecular sieve, and right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining result; E: Left image shows the trajectory of GluN1 / Glu3A protein through a molecular sieve, and right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining result; F: Left image shows the trajectory of GluN1 / Glu3B protein through a molecular sieve, and right image shows the corresponding SDS-PAGE Coomassie Brilliant Blue staining result.

[0196] Figure 5 The results show: A: Live-cell immunofluorescence staining for subunit specificity against Glu2A antibody 2-8C; B: Live-cell immunofluorescence staining for subunit specificity against Glu2B antibody B9; C: Live-cell immunofluorescence staining for subunit specificity against Glu2C antibody 5D5; D: Live-cell immunofluorescence staining for subunit specificity against Glu2D antibody 4D5; E: Live-cell immunofluorescence staining for subunit specificity against Glu3A antibody 1G4; F: Live-cell immunofluorescence staining for subunit specificity against Glu3B antibody 4C9.

[0197] Figure 6The images show: A: Live-cell immunofluorescence staining results of the specific binding sites of anti-Glu2A antibodies 1-5C and 2-8C; B: FSEC detection results of anti-Glu2A antibodies 1-5C and GluN1 / GluN2A proteins; C: Cryo-electron microscopy three-dimensional reconstruction results of 2-8C Fab and GluN1 / GluN2A / GluN3A proteins; D: FSEC detection results of anti-Glu2C antibody 5D5 and GluN1 / GluN2C proteins; E: Cryo-electron microscopy two-dimensional reconstruction results of 5D5 Fab and GluN1 / GluN2A / GluN2C proteins; F: FSEC detection results of anti-Glu2D antibody and GluN1 / GluN2D proteins; G: Cryo-electron microscopy two-dimensional reconstruction results of 4D5, 5E9 Fab and GluN1 / GluN2D proteins. Detailed Implementation

[0198] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time autoantibodies that specifically bind to the GluN1, GluN2, or GluN3 subunits of the NMDA receptor. The anti-NMDA receptor antibodies of this invention can be used for molecular diagnostics or detection, such as the diagnosis, prognosis, and disease monitoring of medical conditions related to NMDAR autoantibodies, and anti-NMDAR encephalitis. In particular, they can serve as competitive NMDA receptor blocking antibodies, specifically blocking the binding of pathogenic antibodies to the NMDA receptor. This invention was completed based on this discovery.

[0199] the term

[0200] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0201] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0202] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0203] Antibody

[0204] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0205] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0206] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0207] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0208] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0209] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0210] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0211] In this invention, the antibody also includes its conserved variants, which are those containing up to 10 amino acid variants, preferably up to 8, and more preferably up to 5, compared to the amino acid sequence of the antibody of this invention.

[0212] Ideally, up to three amino acids are replaced with amino acids of similar or related properties to form a polypeptide. These conserved variant polypeptides are best produced by amino acid substitutions according to Table 1.

[0213] Table A

[0214]

[0215]

[0216] NMDA receptor

[0217] The N-methyl-D-aspartate receptor (NMDA receptor) is an ionotropic glutamate receptor composed of two GLUN1 subunits and two GLUN2 subunits (or two GLUN3 subunits).

[0218] The NMDA receptor has eight splice variants of the GluN1 subtype and two splice variants of the GluN3 subunit (A, B), which can bind to glycine; and four GluN2 subunits (A–D), which can bind to glutamate. The GluN subunits contain two large extracellular domains: an N-terminal domain (ATD) and S1 and S2 domains, the S1 and S2 domains together forming the ligand-binding domain; three transmembrane domains (TM1, 3, 4); an intracellular polypeptide loop (TM2); and an intracellular C-terminal domain that connects to scaffold proteins and messenger systems. The NMDA receptor is crucial for learning and memory, while impaired receptor function is closely associated with conditions such as schizophrenia.

[0219] Nucleic acid

[0220] The present invention also provides a nucleic acid that encodes the heavy chain variable region or light chain variable region of the aforementioned antibody (e.g., an antibody against the NMDA receptor) or recombinant protein or antibody against the NMDA receptor.

[0221] The method for preparing the nucleic acid is a conventional method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis.

[0222] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the protein sequence, while maintaining antibody activity.

[0223] carrier

[0224] The present invention also provides a recombinant expression vector containing the nucleic acid.

[0225] The recombinant expression vector described herein can be obtained by conventional methods in the art, namely, by linking the nucleic acid molecule described in this invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. Preferably, the vector includes various plasmids, granules, bacteriophages, or viral vectors, etc.

[0226] The present invention also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0227] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any common host cells in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells (expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0228] Antibody preparation

[0229] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0230] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0231] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0232] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0233] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0234] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0235] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0236] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0237] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0238] Antibody-drug conjugates (ADCs)

[0239] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention or their antigen-binding fragments.

[0240] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0241] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0242] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0243] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0244] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0245] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0246] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0247] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0248] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0249] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0250] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0251]

[0252] in:

[0253] Ab is an antibody or its antigen-binding fragment.

[0254] LU stands for connector;

[0255] D is a drug;

[0256] Furthermore, the subscript p is a value selected from 1 to 8.

[0257] application

[0258] The present invention also provides the use of the antibodies of the present invention or their antigen-binding fragments, antibody-drug conjugates (ADCs), recombinant proteins, and / or immune cells, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0259] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal NMDA receptor expression or function.

[0260] In this invention, the NMDA receptor-related diseases are those conventionally associated with abnormal NMDA receptor expression or function in the art. Preferably, the diseases associated with abnormal NMDA receptor expression or function include: autoimmune diseases, depression, Alzheimer's disease, schizophrenia, autism, and epilepsy, wherein the autoimmune disease is an autoimmune encephalopathy, preferably autoimmune encephalitis.

[0261] Preferably, the antibodies of the present invention can be used for the development of therapeutic molecules such as blocking antibody binding fragments. For example, the Fab corresponding to the antibody of the present invention is expected to block the binding of autoantibodies to NMDA receptors.

[0262] Pharmaceutical Composition

[0263] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0264] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0265] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0266] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal NMDA receptor expression or function.

[0267] The pharmaceutical compositions of the present invention can be directly used to bind to NMDA receptor protein molecules, and therefore can be used for the prevention and treatment of diseases such as tumors.

[0268] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0269] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0270] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0271] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0272] This invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal NMDA receptor expression or function. Preferably, the disease associated with abnormal NMDA receptor expression or function is a tumor / cancer.

[0273] The main advantages of this invention include

[0274] (1) The present invention prepared an anti-NMDAR antibody that specifically binds to the GluN1 subunit, GluN2A subunit, GluN2B subunit, GluN2C subunit, GluN2D subunit, GluN3A subunit, and GluN3B subunit, and revealed the important binding epitopes of the antibody by immunofluorescence staining and cryo-electron microscopy.

[0275] (2) The antibodies against NMDAR GluN1 subunit, GluN2A subunit and GluN2B subunit of the present invention have the potential to be used to specifically block the binding of NMDAR to autoantibodies in patients with anti-NMDAR encephalitis, thereby achieving the effect of treating anti-NMDAR autoimmune encephalitis.

[0276] (3) The antibodies against NMDAR GluN2A subunit, GluN2B subunit, GluN2C subunit, GluN2D subunit, GluN3A subunit, and GluN3B subunit of the present invention specifically bind to the corresponding subunits and do not cross-react with other subunits, and can be used in scenarios where it is necessary to specifically distinguish different subunits.

[0277] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0278] Example 1: Preparation of GluN1 antibody

[0279] (I) Expression and purification of human GluN1 / GluN3 AMNDA receptor

[0280] 1. Construction of the expression carrier

[0281] To increase protein stability, the intracellular segment sequence of the GluN1 / GluN3A receptor protein was removed. Homologous recombination was used to add expression sequences for the 3C protease, eGFP, and strep tag to the ends of the GluN1 and GluN3A cDNAs, which were then cloned into the pEG-Bacmam expression vector. Following Invitrogen's [specific instructions / method], [followed the instructions / method]. The System method is used to create baculoviruses.

[0282] 2. Protein expression

[0283] Suspended HEK293S GnTI– cells were cultured at 37°C in an incubator containing 5% CO2 until a density of 3.0 × 10⁻⁶ cells was reached. 6 After adding cells per milliliter, baculovirus was added. Twelve hours after infection, sodium butyrate was added to a final concentration of 10 mmol, and the cells were transferred to a 30°C incubator containing 5% carbon dioxide. The cells were then cultured for another 48 hours before harvesting.

[0284] 3. Protein purification

[0285] Cells were resuspended in TBS buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8.0), and then sonicated after adding protease inhibitors (0.8 μM aprotinin, 2 mM pepsin A, 2 μg / ml leuprolide, and 1 mM benzyl sulfonyl fluoride). A final concentration of 1% neopentyl dodecyl maltose glycol (L-MNG), 2 mM cholesterol succinate monoester (CHS), 1 mM glutamate, and 1 mM glycine were added. After 1.5 hours of rotation at 4 degrees Celsius, the cells were centrifuged at 40,000 g for 1 hour, and the supernatant was collected. The supernatant was mixed with Strep-tactin affinity beads and rotated at 4 degrees Celsius for half an hour. The beads were washed with washing buffer (1% L-MNG, 2 mM CHS in TBS buffer), and then the proteins were eluted with elution buffer (washing buffer with 5 mM dethiobiotin added). After overnight digestion with 4°C 3C protease, the corresponding tetrameric protein was further purified using GE's Superose 6 10 / 300GL molecular sieve. Figure 1 We then collected the purified tetrameric NMDA receptor and stained the protein with Coomassie Brilliant Blue after SDS-page staining. The staining results showed two clear bands, with the band positions corresponding to the molecular weights of GluN1 and GluN3A, respectively, indicating that there were no other contaminating proteins in the purified protein. Figure 1 ).

[0286] (II) Animal Immunization

[0287] The purified protein was used as an antigen to immunize mice. The specific immunization procedure was as follows: the protein and manganese adjuvant were mixed at a mass ratio of 1:5 and then used to immunize the mice. Immunization was performed every 10 days for a total of 3 times. Each mouse received 100ug for the first immunization, and 50ug for the second and third immunizations. One week after the third immunization, a booster immunization of 60ug per mouse was performed. Three days after the booster immunization, spleen cells from the mice were fused with myeloma cells Sp2 / 0 for cell fusion.

[0288] (III) Screening and Identification of Positive Clones

[0289] After cell fusion, live-cell immunofluorescence was used to screen for positive hybridoma cell lines specifically targeting GluN1, and three subcloning processes were performed to obtain the corresponding monoclonal antibodies. The hybridoma cells were then injected into mice to prepare ascites fluid, which was collected and purified to obtain the corresponding antibodies. After screening, we obtained:

[0290] Antibodies specifically targeting GluN1 include 4F11, 5E10, 1B6, 4E8, and 5C10 (staining results for representative antibody 4F11 are shown in [reference]). Figure 2 ),

[0291] The results showed that 4F11 could bind to GluN1 / GluN2A, GluN1 / GluN2B, GluN1 / GluN2C, GluN1 / GluN2D, GluN1 / GluN3A, and GluN1 / GluN3B.

[0292] 4F11 was confirmed to be an antibody that specifically recognizes GluN1.

[0293] In addition, N1 is not bound to the LBD, while N2A is bound to the LBD.

[0294] Example 2: Analysis of the GluN1 antibody binding epitope

[0295] Meanwhile, the function of 4F11 was further verified using FSEC and cryo-electron microscopy. FSEC results showed that the addition of GluN1 Fab to GluN1 / GluN2A, GluN1 / GluN2B, GluN1 / GluN2C, and GluN1 / GluN2D proteins respectively resulted in varying degrees of leftward shift in the peak position compared to the individual GluN1 / GluN2A, GluN1 / GluN2B, GluN1 / GluN2C, and GluN1 / GluN2D proteins. This is presumably due to the larger molecular weight of the complex formed after Fab binds to the protein, thus shifting the peak position to the left. Figure 3 );

[0296] To determine the specific binding site of 4F11, the purified GluN1 / GluN2A-4F11 Fab complex was placed on a dense copper mesh, and the liquid was absorbed into a thin layer using filter paper. The mixture was then flash-frozen in liquid ethane to momentarily hold the protein at different angles, followed by liquid nitrogen preservation. Images of the sample were acquired using a 120 kV electron microscope, yielding two-dimensional images of the protein from different angles. A three-dimensional reconstruction of the protein structure was then performed using a computer, ultimately analyzing the three-dimensional electron density maps of the protein in different states.

[0297] The cryo-electron microscopy structure analysis shows that 4F11 is bound to the NTD of the GluN1 subunit. Figure 3 ).

[0298] Example 3: Preparation of anti-GluN2 and anti-GluN3 antibodies

[0299] (I) Expression and purification of human GluN1 / GluN2A, GluN1 / GluN2B, GluN1 / GluN2C, GluN1 / GluN2D, GluN1 / GluN3A, and GluN1 / GluN3B subtype NMDA receptors

[0300] 1. Construction of the expression carrier

[0301] To increase protein stability, the intracellular sequences of the GluN1 / GluN2A, GluN1 / GluN2B, GluN1 / GluN2C, GluN1 / GluN2D, GluN1 / GluN3A, and GluN1 / GluN3B receptor proteins were removed. Homologous recombination was used to add expression sequences for 3C protease, eGFP (with GluN1 / GluN2C fused to mRuby), and a strep tag to the ends of the cDNAs of GluN1 and GluN2A, and then cloned them into the pEG-Bacmam expression vector. Following Invitrogen's [manufacturer's] instructions... The System method is used to create baculoviruses.

[0302] 2. Protein expression

[0303] The suspended HEK293S GnTI – Cells were cultured at a density of 3.0 × 10⁶ cells / year in a 37°C incubator containing 5% carbon dioxide until the density reached 3.0 × 10⁶ cells / year. 6 After adding cells per milliliter, baculovirus was added. Twelve hours after infection, sodium butyrate was added to a final concentration of 10 mmol, and the cells were transferred to a 30°C incubator containing 5% carbon dioxide. The cells were then cultured for another 48 hours before harvesting.

[0304] 3. Protein purification

[0305] Cells were resuspended in TBS buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8.0), and then sonicated after adding protease inhibitors (0.8 μM aprotinin, 2 mM pepsin A, 2 μg / ml leuprolide, and 1 mM benzyl sulfonyl fluoride). A final concentration of 1% neopentyl dodecyl maltose glycol (L-MNG), 2 mM cholesterol succinate monoester (CHS), 1 mM glutamate, and 1 mM glycine were added. After 1.5 hours of rotation at 4 degrees Celsius, the cells were centrifuged at 40,000 g for 1 hour, and the supernatant was collected. The supernatant was mixed with Strep-tactin affinity beads and rotated at 4 degrees Celsius for half an hour. The beads were washed with washing buffer (1% L-MNG, 2 mM CHS in TBS buffer), and then the proteins were eluted with elution buffer (washing buffer with 5 mM dethiobiotin added). After overnight digestion with 4°C 3C protease, the corresponding tetrameric protein was further purified using GE's Superose 6 10 / 300GL molecular sieve. Figure 4 We then collected the purified tetrameric NMDA receptor and stained the protein with Coomassie Brilliant Blue after SDS-page staining. The staining results showed two clear bands, with the band positions corresponding to the molecular weights of GluN1 and GluN2 (GluN3), indicating that there were no other contaminating proteins in the purified protein. Figure 4 ).

[0306] (II) Animal Immunization

[0307] The purified protein was used as an antigen to immunize mice. The specific immunization procedure was as follows: the protein and manganese adjuvant were mixed at a mass ratio of 1:5 and then used to immunize the mice. Immunization was performed every 10 days for a total of 3 times. Each mouse received 100ug for the first immunization, and 50ug for the second and third immunizations. One week after the third immunization, a booster immunization of 60ug per mouse was performed. Three days after the booster immunization, spleen cells from the mice were fused with myeloma cells Sp2 / 0 for cell fusion.

[0308] (III) Screening and Identification of Positive Clones

[0309] After cell fusion, live-cell immunofluorescence was used to screen for positive hybridoma cell lines specifically targeting GluN2 or GluN3 (depending on the immunogen: GluN1 / Glu2A for Glu2A antibodies; GluN1 / Glu2B for Glu2B antibodies; GluN1 / Glu2C for Glu2C antibodies; GluN1 / Glu2D for Glu2D antibodies; GluN1 / Glu3A for Glu3A antibodies; GluN1 / Glu3B for Glu3B antibodies);). Three subcloning operations were then performed to obtain the corresponding monoclonal antibodies. The hybridoma cells were then injected into mice to prepare ascites fluid, which was collected and purified to obtain the corresponding antibodies. After screening, we obtained:

[0310] Specific antibodies against Glu2A, 1-5C and 2-8C (representative antibody 2-8C staining results are shown in [reference]). Figure 5 (A)

[0311] Antibody B9 specifically targeting Glu2B (staining results are shown in...) Figure 5 (B)

[0312] Antibody 5D5 specifically targeting Glu2C (staining results are shown in...) Figure 5 (C)

[0313] Antibodies specifically targeting Glu2D, 1C10, 4D5, and 5E9 (representative antibody 4D5 staining results are shown in [reference]). Figure 5 D),

[0314] Antibodies specifically targeting Glu3A, 3E, 1G4, and 1G9 (staining results of representative antibody 1G4 are shown in [reference]). Figure 5 (E),

[0315] Antibodies 4C9 and 6D12 specifically targeting Glu3B (representative antibody 4C9 staining results are shown in...) Figure 5 (F).

[0316] Example 3: Analysis of the binding epitopes of anti-GluN2 and anti-GluN3 antibodies

[0317] Meanwhile, the specific functions targeting GluN2 and GluN3 were further verified using live-cell immunofluorescence staining, FSEC, and cryo-electron microscopy with chimeric plasmids.

[0318] Live cell staining was performed on cells transfected with two anti-Glu2A antibodies and a chimeric plasmid of GluN1 / GluN2D to verify the results. It was observed that 1-5C can bind to the GluN1 / GluN2D protein with the NTD of 2D replaced by the NTD of 2A, proving that 1-5C binds to the NTD of GluN2A. Figure 6 (A); while 2-8C can bind to the GluN1 / GluN2D protein that replaces 2D LBD-S1 with 2A LBD-S1, proving that 2-8C binds to LBD-S1 of GluN2A ( Figure 6 (A), this result was also confirmed by cryo-electron microscopy (cryo-electron microscopy). Figure 6 (C)

[0319] FSEC results showed that the addition of Fab, an anti-GluN2A-NTD inhibitor, to the GluN1 / GluN2A protein shifted the peak position to the left compared to the GluN1 / GluN2A protein alone. This is presumably because the complex formed by Fab and the protein has a larger molecular weight, thus shifting the peak position to the left. Figure 6 (B)

[0320] Adding Fab, an anti-GluN2C protein, to the GluN1 / GluN2C protein resulted in a leftward shift in the protein's elution position compared to the GluN1 / GluN2C protein alone. Figure 6 (D).

[0321] To determine the specific binding site of the anti-GluN2C antibody 5D5, the purified GluN1 / GluN2A / GluN2C-5D5 Fab complex was placed on a dense copper mesh, and the liquid was absorbed into a thin layer using filter paper. The mixture was then flash-frozen in liquid ethane to momentarily hold the protein at different angles, followed by liquid nitrogen preservation. Images of the sample were acquired using a 120 kV electron microscope, yielding two-dimensional images of the protein from different angles. The protein structure was then reconstructed using a computer.

[0322] The cryo-electron microscopy structure analysis shows that 5D5 is bound to the NTD of the Glu2C subunit. Figure 6 (E).

[0323] After adding the anti-GluN2D Fab to the GluN1 / GluN2D protein, the elution position of the protein showed varying degrees of leftward shift compared to the GluN1 / GluN2D protein alone. Figure 6 (F)

[0324] To determine the specific binding sites of the anti-GluN2D antibody 4D5 and 5E9, purified GluN1 / GluN2D-4D5 and GluN1 / GluN2D-5E9 Fab complexes were subjected to 120 kV cryo-electron microscopy data collection. Two-dimensional reconstruction revealed the specific binding mode between the antibody and the protein; both 4D5 and 5E9 were bound to the NTD of GluN2D. Figure 6 (G).

[0325] discuss:

[0326] NMDA receptors are crucial for learning and memory, and receptor dysfunction is associated with many neuropsychiatric disorders such as depression, Alzheimer's disease, schizophrenia, and autism. Anti-NMDA receptor encephalitis occurs because patients produce high titers of IgG-type autoantibodies against the GluN1, Glu2A, or Glu2B subunits.

[0327] The Fab fragments of antibodies specifically targeting GluN1, Glu2A, and Glu2B produced in this invention have the potential to serve as a "drug" that specifically blocks the binding of antibodies to NMDAR in encephalitis patients, thus playing a positive role in the treatment of NMDAR encephalitis. Furthermore, in clinical diagnosis, the current clinical diagnosis of anti-NMDA receptor encephalitis mainly relies on the detection of anti-NMDA receptor antibodies in the patient's serum and cerebrospinal fluid. Antibodies against GluN1, Glu2A, and Glu2B can be used as standards in the detection process to double-validate the accuracy of clinical sample testing.

[0328] NMDAR receptors form tetramers in vivo to perform their physiological functions. Regardless of whether they form di- or tri-tetramers, it is difficult to distinguish the corresponding subunits. However, the antibody produced by this invention is highly specific and does not cross-react with other subunits, which can distinguish the specific subunits.

[0329] Antibody variable region sequence information:

[0330] (N1)4F11-VH SEQ ID NO:1

[0331] DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YISYSGSTGYNPSLKS RISITRDTSKNQFFLQLNSVTTEDTATYYCAR LYYGFYGMDY WGQGTSVTVSSAKTT

[0332] 4F11-VK:

[0333] DIVLTQSPASLAVSLGQRATISC KASQSVDYDGNSYMN WYQQKPGQPPKLLIY AASNLES GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQGNEDPPT FGGGTKLEIK

[0334] (2A)1-5C-VH:SEQ ID NO:2

[0335] LQESGGGLVKPGGSLKLSCAASGFAFS NYDMS WVRQTPEKRLEWVA SISNGGSYTYYPDSMKG RFTISRDNARNTLYLQMSSLRSEDTALYYCAR PFFLRLRRTWFAY WGQGTLVTVS

[0336] 1-5C-VK:

[0337] IVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKTYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPLT FGAGTKLEL

[0338] (2B)B9-VH:SEQ ID NO:3

[0339] GVQLQESGGASVKPGGSLKLSCAASGFDFS SYDMS WVRQTPEKRLEWVA YISSGGGNTYYPDTVKG RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAT RRDFYFDY WGQGTTLTVSSAK

[0340] B9-VK:SEQ ID NO:4

[0341] DIVLTQSPASLAVSLGQRATISC RASQSVSTSKYSYMH WYQQKPGQPPKLLIK YASNLES GVPARFSGSGSGTDFTLNIHPVEEEDTATYFC QHSWKIPYT FGGGTKLEIK

[0342] (2C)5D5-VH:SEQ ID NO:5

[0343] DVQLQESGPDLVKPSQSLSLTCTVTGYSIT SAYSWH WIRQFPGNKLEWMG YIYYSGSTNYNPFLKS RISITRDTSKNQFFLQLNSVTTEDTATYYCTT YYYGSGFAY WGQGTLVTVSAAK

[0344] 5D5-VK:SEQ ID NO:6

[0345] DIKMTQSPSSMYTSLGERVTITC KASQDIDNYLSWFQQKPGKSPKTLIY RANRLID GVPSRFSGSGSGQDFSLTISSLEYEDMAIYYC LQYDEFPFT FGSGTKLEIK

[0346] (2D)5E9-VH:SEQ ID NO:41

[0347] EVMLVESGGGLVKPGGSLKLSCAASGFTFS SYAMS WVRQTPEKRLEWVA TISSGGSYTYYPDSVKG RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR QVRYYFDY WGQGTTLTVSSAK

[0348] 5E9-VK:SEQ ID NO:42

[0349] QITQSPSYLAASPGETITINC RASKSIGKYLA WYQEKPGKTYKLLIY SGSTLQS GFPSRFSGSGSGTDFTLTISSLEPEDFAMYYC QQHNEYPFT FGAGTKLELK

[0350] (3A)3E-VH:SEQ ID NO:7

[0351] QVTLKESGPGILRPSQTLSLTCSFSGFSLS TSGMGVG WIRQPSGKGLEWLA HIWWDDVKRYNPALKS RLTISKNTSSSQVFLKIASVDTADTATYCAR ITEDIEGYDEWFAY WGQGTLVTVSAAK

[0352] 3E-VK:SEQ ID NO:8

[0353] DIQMSQSPSSLAVSVGEKVTMSC KSSQSLLYSRNQKNYLA WYQQKPGQSPKLLIY WASTRES GVPDRFTGSGSGTDFLTLTISSVKAEDLAVYYC QQYYSYPFT FGSGTKLEIK

[0354] 4F11-VH:

[0355] HCDR1 SDYAWN SEQ ID NO:11

[0356] HCDR2 YISYSGSTGYNPSLKS SEQ ID NO:12

[0357] HCDR3 LYYGFYGMDY SEQ ID NO:13

[0358] 4F11-VK:

[0359] LCDR1 KASQSVDYDGNSYMN SEQ ID NO:14

[0360] LCDR2 AASNLES SEQ ID NO:15

[0361] LCDR3 QQGNEDPPT SEQ ID NO:16

[0362] 1-5C-VH:

[0363] HCDR1 NYDMS SEQ ID NO:17

[0364] HCDR2 SISNGGSYTYYPDSMKG SEQ ID NO:18

[0365] HCDR3 PFFLRLRRTWFAY SEQ ID NO:19

[0366] 1-5C-VK:

[0367] LCDR1 KSSQSLLNSGNQKTYLT SEQ ID NO:20

[0368] LCDR2 WASTRES SEQ ID NO:21

[0369] LCDR3 QNDYSYPLT SEQ ID NO:22

[0370] B9-VH:

[0371] HCDR1 SYDMS SEQ ID NO:23

[0372] HCDR2 YISSGGGNTYYPDTVKG SEQ ID NO:24

[0373] HCDR3 RRDFYFDY SEQ ID NO:25

[0374] B9-VK:

[0375] LCDR1 RASQSVSTSKYSYMH SEQ ID NO:26

[0376] LCDR2 YASNLES SEQ ID NO:27

[0377] LCDR3 QHSWKIPYT SEQ ID NO:28

[0378] 5D5-VH:

[0379] HCDR1 SAYSWH SEQ ID NO:29

[0380] HCDR2 YIYYSGSTNYNPFLKS SEQ ID NO:30

[0381] HCDR3 YYYGSGFAY SEQ ID NO:31

[0382] 5D5-VK:

[0383] LCDR1 KASQDIDNYL SEQ ID NO:32

[0384] LCDR2 RANRLID SEQ ID NO:33

[0385] LCDR3 LQYDEFPFT SEQ ID NO:34

[0386] 3E-VH:

[0387] HCDR1 TSGMGVG SEQ ID NO:35

[0388] HCDR2 HIWWDDVKRYNPALKS SEQ ID NO:36

[0389] HCDR3 ITEDIEGYDEWFAY SEQ ID NO:37

[0390] 3E-VK:

[0391] LCDR1 KSSQSLLYSRNQKNYLA SEQ ID NO:38

[0392] LCDR2 WASTRES SEQ ID NO:39

[0393] LCDR3 QQYYSYPFT SEQ ID NO:40

[0394] 5E9-VH:

[0395] HCDR1 SYAMS SEQ ID NO:43

[0396] HCDR2 TISSGGSYTYYPDSVKG SEQ ID NO:44

[0397] HCDR3 QVRYYFDY SEQ ID NO:45

[0398] 5E9-VK:

[0399] LCDR1 RASKSIGKYLA SEQ ID NO:46

[0400] LCDR2 SGSTLQS SEQ ID NO:47

[0401] LCDR3 QQHNEYPFT SEQ ID NO:48

[0402] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A heavy chain variable region of an antibody, characterized in that, The heavy chain variable region includes the following three complementary determinant regions (CDRs): HCDR1, as shown in SEQ ID NO:11, HCDR2 shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; or HCDR1, as shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:18, and HCDR3 as shown in SEQ ID NO:19; or HCDR1, as shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:25; or HCDR1, as shown in SEQ ID NO:29, HCDR2 shown in SEQ ID NO:30, and HCDR3 as shown in SEQ ID NO:31; or HCDR1, as shown in SEQ ID NO:35, HCDR2 shown in SEQ ID NO:36, and HCDR3 as shown in SEQ ID NO:37; or HCDR1, as shown in SEQ ID NO:43, HCDR2 shown in SEQ ID NO:44, and HCDR3 as shown in SEQ ID NO:45; Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

2. A heavy chain of an antibody, characterized in that, The heavy chain has the heavy chain variable region and heavy chain constant region as described in claim 1.

3. A light chain variable region of an antibody, characterized in that, The light chain variable region has a complementary determinant region (CDR) selected from the following group: LCDR1 shown in SEQ ID NO:14 LCDR2 shown in SEQ ID NO:15, and LCDR3 shown in SEQ ID NO:16; or LCDR1 shown in SEQ ID NO:20 LCDR2 shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:22; or LCDR1 shown in SEQ ID NO:23, LCDR2 shown in SEQ ID NO:26, and LCDR3 shown in SEQ ID NO:27; or LCDR1 shown in SEQ ID NO:28 LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; or LCDR1 shown in SEQ ID NO:38 LCDR2 shown in SEQ ID NO:39, and LCDR3 as shown in SEQ ID NO:40; or LCDR1 shown in SEQ ID NO:46 LCDR2 shown in SEQ ID NO:47, and LCDR3 as shown in SEQ ID NO:48; Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

4. A light chain of an antibody, characterized in that, The light chain has the light chain variable region and light chain constant region as described in claim 3.

5. An antibody or its antigen-binding fragment, characterized in that, The antibody has the following characteristics: (1) The heavy chain variable region as described in claim 1; and / or (2) The light chain variable region as described in claim 3; Preferably, the antibody has: the heavy chain as described in claim 2; and / or the light chain as described in claim 4; Among them, any of the above amino acid sequences may also include a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining NMDA receptor binding affinity.

6. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the sequence of the heavy chain variable region as claimed in claim 1, the sequence of the heavy chain as claimed in claim 2, the sequence of the light chain variable region as claimed in claim 3, the sequence of the light chain as claimed in claim 4, or the sequence of the antibody or its antigen-binding fragment as claimed in claim 5; and (ii) Optional tag sequences to assist in expression and / or purification.

7. A polynucleotide, characterized in that, It encodes a polypeptide selected from the following group: (1) The heavy chain variable region as described in claim 1, the heavy chain as described in claim 2, the light chain variable region as described in claim 3, the light chain as described in claim 4, or the antibody or its antigen-binding fragment as described in claim 5; or (2) The recombinant protein as described in claim 6.

8. A carrier, characterized in that, It contains the polynucleotide as described in claim 7.

9. A genetically engineered host cell, characterized in that, It contains the vector of claim 8 or the genome in which the polynucleotide of claim 7 is integrated.

10. An immunoconjugate, characterized in that, This immunoconjugate contains: (a) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody or antigen-binding fragment thereof of claim 5, and the recombinant protein of claim 6; and (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

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