ANTIBODY OR ANTIBODY FRAGMENT OF NON-IG SCAFFOLD BINDING TO A BINDING REGION OF AN ANTI-N-METHYL-D-ASPARTATE (NMDA) RECEPTOR ANTIBODY

Abstract

Subject matter of the present invention is an Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody and its uses in therapy or diagnosis.

Claims

1. Antibody or antibody fragment or non-Ig scaffold binding specifically to a binding region of an anti-NMDAR1 antibody wherein the binding region of said anti-NMDAR1 antibody is comprised in one ore more sequences wherein said one or more sequence is selected from a group consisting of the following sequences: TABLE-US-00009 (003-109-HC) SEQIDNO:1 VQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV IWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR HYDFDAFDIWGQGTMVTVSS (003-109-LC) SEQIDNO:2 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLM IYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSST LYVFGTGTKVTVL (003-102-HC) SEQIDNO:3 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWI GEIYHSGNTNYNPSLKSRVTVSVDKSKNQFSLKLTSVTAADTAVYYCAR DVSGGVNWFDPWGQGTLVTVSS (003-102-LC) SEQIDNO:4 NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVI YEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSST VVFGGGTKLTVL (007-168-HC) SEQIDNO:5 VQLVQSGAEAKKPGESLKISCKASGYSFTTFWIGWVRQMPGSGLEWIGI IYPGDSDTRYSPSFQGHVTISADRSTSTAYLQWSSLKASDTAMYYCARS AVFDYWGQGTLVTVSS (007-168-LC) SEQIDNO:6 EIVMTQSPATLSVSPGGRATLSCRASQSVSSNLAWYQQKPGQAPRLLIY GASTRATGIPVRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPTSW TFGQGTKVEIK (007-169-HC) SEQIDNO:7 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMG IIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR DYGDYYFDYWGQGTLVTVSS (007-169-LC) SEQIDNO:8 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLM IYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYT GVFGEGTKLTVL (007-124-HC) SEQIDNO:9 EVQLVESGGGVGRPGGSLRLSCAASGFTFDDYGMSWVRQVPGKGLEWVS GINWSGADTGYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYHCAR EVGIAVTGYWFDPWGQGTLVTV (007-124-LC) SEQIDNO:10 SYELTQPPSVSVAPGQTARISCGGNHSESVHWYQQKPGQAPVLVVYDDS DRPSGIPERFSGSKSGNTATLTISRVEGGDEAEYYCQVWDSSSDHPGVV FGGGTKLTVL (007-142-HC) SEQIDNO:11 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMG IIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR DYGDYYFDYWGQGTLVTVSS (007-142-LC) SEQIDNO:12 LTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYD VSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTGVF GGGTKLTVL (008-218-HC) SEQIDNO:13 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVS GISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DRASSWYAYGMDVWGQGTLVTV (008-218-LC) SEQIDNO:14 NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVI YDDNQRPSGVPNRFSGSIDSSSNSASLIISGLKTEDEADYYCQSTRVFG GGTKLTVL and wherein said binding region for the antibody or antibody fragment or non-Ig scaffold comprises or consists of one or several of the below mentioned sequences: TABLE-US-00010 SEQIDNO:15 GFTFSSYG SEQIDNO:16 IWYDGSNK SEQIDNO:17 ARRHYDFDAFDI SEQIDNO:18 SSDVGGYNY SEQIDNO:19 EVS SEQIDNO:20 SSYTSSSTLYV SEQIDNO:21 GGSISSSNW SEQIDNO:22 IYHSGNT SEQIDNO:23 ARDVSGGVNWFDP SEQIDNO:24 SGSIASNY SEQIDNO:25 EDN SEQIDNO:26 QSYDSSTVV SEQIDNO:27 GYSFTTFW SEQIDNO:28 IYPGDSDT SEQIDNO:29 ARSAVFDY SEQIDNO:30 QSVSSN SEQIDNO:31 GAS SEQIDNO:32 QQYNNWPTSWT SEQIDNO:33 GYSFTSYW SEQIDNO:34 IYPGDSD SEQIDNO:35 ARDYGDYYFDY SEQIDNO:36 SSDVGGYNY SEQIDNO:37 DVS SEQIDNO:38 CSYAGSYTGV SEQIDNO:39 GFTFDDYG SEQIDNO:40 INWSGADT SEQIDNO:41 AREVGIAVTGYWFDP SEQIDNO:42 HSES SEQIDNO:43 DDS SEQIDNO:44 QVWDSSSDHPGVV SEQIDNO:45 GYSFTSYW SEQIDNO:46 IYPGDSDT SEQIDNO:47 ARDYGDYYFDY SEQIDNO:48 SSDVGGYNY SEQIDNO:49 DVS SEQIDNO:50 CSYAGSYTGV SEQIDNO:51 GFTFDDYA SEQIDNO:52 ISWSSGSI SEQIDNO:53 AKDRASSWYAYGMDV SEQIDNO:54 SGSIASNY SEQIDNO:55 DDN SEQIDNO:56 QSTRV

2. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 wherein said antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody is a non-IgG scaffold.

3. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 wherein said non-Ig scaffold may be selected from the group comprising tetranectin-based non-Ig scaffold, fibronectin scaffold, lipocalin-based scaffold, ubiquitin scaffolds, transferrin scaffolds, protein A scaffolds, ankyrin repeat based scaffolds, microproteins, preferably microproteins forming a cysteine knot, scaffolds, Fyn SH3 domain based scaffolds, EGFR-A-domain based scaffolds and Kunitz domain based scaffolds and aptamers.

4. Non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 3 wherein said non-Ig scaffold is an oligonucleotide aptamer.

5. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according claim 1 wherein said antibody or antibody fragment or non-Ig scaffold exhibits an affinity towards said binding region of an anti-NMDAR1 antibody in such that the dissociation constant KD is lower than 10-7 M, preferred 10-8 M, preferred KD is lower than 10-9 M, most preferred lower than 10-10 M. to said binding region of the anti-NMDAR1 antibody.

6. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 wherein said antibody or antibody fragment or non-Ig scaffold neutralizes the anti-NMDAR1 antibodies.

7. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 wherein said antibody or antibody fragment or non-Ig scaffold is a monospecific antibody or antibody fragment or non-Ig scaffold.

8. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition in a subject in need of such a therapy said disease or condition being associated with anti-NMDAR1 antibodies.

9. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition said disease or condition being associated with anti-NMDAR1 antibodies in a subject in need if such a therapy wherein a subject is in need of such a therapy if said subject exhibits the presence of anti-NMDAR1 antibodies in a bodily fluid and exhibits at least one clinical symptom or clinical condition selected from the group comprising the clinical symptoms/conditions according to the following list (ICD numbers in parentheses refer to the WHO International Classification of Diseases which defines the clinical conditions): psychiatric abnormalities including depression (F32), mania with psychotic symptoms (F30.2), anxiety (F06.4), phobic anxiety (F40), delusions (F22.0), obsessive-compulsive disorder (F42), organic delusional disorder (F06.3), catatonia (F06.1, F20.2), acute polymorphic psychotic disorder (F23.0, F23.1), dissociative disorders (F44) movement disorders including dyskinesias/dystonia (G24), myoclonus (G25.3), tremor (G25.0, G25-1, G25-2), tics (F95, G25.69) epileptic seizures (G40) hypoventilation (R06.89) mild cognitive impairment (F06.7) dementia in Alzheimer's disease (F00), vascular dementia (F01), dementia in other diseases (F02) pregnancy.

10. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition in a subject said disease or condition being associated with anti-NMDAR1 antibodies wherein said antibody or antibody fragment or non-Ig scaffold is administered in vivo to said subject in need thereof.

11. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition in a subject said disease or condition being associated with anti-NMDAR1 antibodies wherein said antibody or antibody fragment or non-Ig scaffold is administered to said subject in need thereof intravenously.

12. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition in a subject said disease or condition being associated with anti-NMDAR1 antibodies said antibody or antibody fragment or non-Ig scaffold is used in an ex vivo therapy of said subject in need thereof.

13. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition in a subject said disease or condition being associated with anti-NMDAR1 antibodies wherein said subject exhibits the presence of anti-NMDAR1 antibodies when measured according to a method of Example 1.2.

14. Antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according to claim 1 for use in therapy of a disease or a condition in a subject said disease or condition being associated with anti-NMDAR1 antibodies wherein said antibody or antibody fragment or non-Ig scaffold is to be used in combination with a chemotherapeutic agent or a immunosuppressive agent.

15. A method of treatment or prevention of a disease or medical condition associated with anti-NMDAR1 antibodies, which comprises the administration of an effective amount of antibody or antibody fragment or non-Ig scaffold binding to a binding region of an anti-NMDAR1 antibody according claim 1 to a subject in need thereof.

16. A medicament for the treatment or prevention of a disease or medical condition associated with anti-NMDAR1 antibodies comprising a compound according to claim 1.

17. Antibody or antibody fragment or non-Ig scaffold-coated device for plasma exchange or CSF exchange (liquorpheresis) wherein said antibody or antibody fragment or non-Ig scaffold is an antibody or antibody fragment or non-Ig scaffold according to claim 1.

18. Antibody or antibody fragment or non-Ig scaffold-coated device for plasma exchange or CSF exchange (liquorpheresis) according to claim 17 wherein said antibody or antibody fragment or non-Ig scaffold-coated device is an antibody or antibody fragment or non-Ig scaffold-coated column.

19. Use of an antibody or antibody fragment or non-Ig scaffold according to claim 1 for determining the presence of anti-NMDAR1 antibodies in a sample of a bodily fluid of a subject having a disease or condition being associated with anti-NMDAR1 antibodies.

20. A method of determining the presence of an anti-NMDAR1 antibody in a sample of bodily fluid of a subject in order to determine whether said subject is in need of a therapy wherein said subject has a disease or condition being associated with anti-NMDAR1 antibodies and the method comprising: contacting a sample of bodily fluid obtained from said subject with at least one antibody or antibody fragment or non-Ig scaffold according to claim 1 determining the presence of said anti-NMDAR1 antibodies in said sample of bodily fluid wherein in case an anti-NMDAR1 antibody is present in said sample said subject may have a disease or condition associated with anti-NMDAR1 antibodies and may be in need of a therapy.

21. Kit for determining the presence of antibodies in sample of a subject that may be in need of a therapy comprising: 1) A solid support with immobilized mixture of NMDAR1-binding antibodies or NMDAR1-binding antibody fragments or NMDAR1-binding non-Ig scaffolds 2) Recombinant human NMDAR1 antibody for determination of standard curve. Anti-human immunoglobulin antibody conjugated to a quantifiable marker

Description

EXAMPLES

Example 1

[0146] 1.1 Generation of Monoclonal Human Recombinant NMDAR1 Antibodies

[0147] (Technical procedure based on Tiller et al. 2009, J Immunol Methods 350(1-2):183-93)

[0148] Isolation of Single Human Plasma Cells and Memory B Cells from Cerebrospinal Fluid samples (FIG. 3-4)

[0149] Cerebrospinal fluid samples (CSF) were collected in the context of the general routine examination after signed informed consent in accordance with Charit ethics board approval. CSF samples were centrifuged at 400g for 10 minutes. Then supernatant was decanted and cells were suspended in 500 l of freezing medium (45% RPMI, 45% FCS, 10% DMSO) to be stored at 80 C. until further use. For the fluorescence activated cell sorting (FACS), frozen cells were thawed, diluted and stained on ice with the antibodies as shown in FIGS. 3-4. Cell sorting was performed on an FACSAria II (BD Biosciences) into 96-well PCR plates (VWR) containing 4 l/well of ice-cold lysis solution of 0.5 phosphate-buffered saline (PBS) with 10 mM DTT (Invitrogen), 8 U RNAsin (Promega). After the sort, plates were directly sealed with Sealing Foil (VWR) and immediately frozen on dry ice before storage at 80 C.

[0150] Single Cell Reverse Transcription PCR and Amplification of Ig Genes

[0151] The reverse transcription (RT) was performed in the original 96-well sorting plate in a total volume of 14 l per sample. To the total RNA from each single cell, to each well 150 ng random hexamer primer p(dN).sub.6 (Roche), 0.5 l of 25 mM from each nucleotide dNTP-Mix (Invitrogen), 1 l 0.1 M DTT (Invitrogen), 0.5 l 10% Igepal CA-630 (Sigma), 14 U RNAsin (Promega) and 50 U Superscript III reverse transcriptase (Invitrogen) were added. Thermal cycling conditions were 42 C. for 10 min, 25 C. for 10 min, 50 C. for 60 min and 94 C. for 5 min. cDNA was stored at 20 C.

[0152] For Ig V gene amplification a nested PCR strategy in two steps was used, for each single cell cDNA separately for IgH, Ig and Ig. All PCR reactions were performed in 96-well plates (VWR) in a total volume of 40 l per well containing 320 nM of total primer or primer mix, 250 nM each dNTP (Invitrogen) and 0.9 U HotStar Taq DNA polymerase (Qiagen). As templates for first PCR's 2.0 l of cDNA were used, for nested reactions 3.5 l of unpurified first PCR product. Each round of PCR was performed at initial 94 C. for 15 min, 50 cycles at 94 C. for 30 sec, 58 C. (IgH/Ig) or 60 C. (Ig) for 30 sec and 72 C. for 55 sec (1st PCR) or 45 sec (2nd PCR) before final 72 C. for 10 min.

[0153] Ig Gene Sequence Analysis

[0154] The second PCR products were sequenced with the respective reverse primer for IgH, Ig bzw. Ig as outlined in Tiller et al. 2009. J Immunol Methods 350(1-2):183-93. Sequences were analyzed by IgBLAST comparison with GenBank (Ye J et al. 2013. Nucleic Acids Res. 41)) to identify germline V(D)J gene segments with highest identity. IgH complementarity determining region (CDR)3 length was determined as indicated in IgBLAST by counting the amino acid residues following framework region (FWR)3 up to the conserved tryptophan-glycine motif in all JH segments or up to the conserved phenylalanin-glycine motif in JL segments. In contrast to sequences from cloned Ig genes, 2nd PCR product sequences are unlikely to show the mutations that were introduced by the Taq polymerase and would do so only if the mutations were introduced early during the PCR. Analysis of Ig gene sequences from naive B cells lacking somatic mutations allows the detection of Taq-mediated misincorporated nucleotides by comparison to published germline sequences.

[0155] Expression Vector Cloning

[0156] Before cloning, all PCR products were purified using Qia-Quick 96 PCR Purification Kit (Qiagen) and QIAvac96. Samples were eluted with 50 l nuclease-free water (Eppendorf) into 96-well plates. Digests were carried out with the respective restriction enzymes AgeI, SalI and XhoI (all from NEB) in the same plate in a total volume of 35-40 l and digested PCR products were purified before ligation into human Ig1, Ig and Ig, expression vectors containing an Ig gene signal peptide sequence (GenBank accession no. DQ407610) and a multiple cloning site upstream of the human Ig1, Ig or Ig, constant regions. Transcription is under the influence of the human cytomegalovirus (HCMV) promotor and clones can be selected based on resistance to ampicillin. Ligation was performed in a total volume of 10 l with 1 U T4-Ligase (Invitrogen), 7.5 l of digested and purified PCR product and 25 ng linearized vector. Competent E. coli DH10B bacteria (Clontech) were transformed at 42 C. with 3 l of the ligation product in 96-well plates. Colonies were screened by PCR using 5 Absense as forward primer and 3 IgGinternal, 3 C494 or 3 C, as reverse primer, respectively. PCR products of the expected size (650 bp for Ig1, 700 bp for Ig and 590 bp for Ig) were sequenced to confirm identity with the original PCR products. (Tiller et al. 2009, J Immunol Methods 350(1-2):183-93). Due to the use of error-prone Taq-Polymerase approximately Plasmid DNA was isolated from 3 ml bacteria cultures grown for 16 h at 37 C. in Terrific Broth (Difco Laboratories) containing 75 g/ml ampicillin (Sigma) using QIAprep Spin columns (Qiagen). From 1.5 ml baceria cultures, on average 35 g plasmid DNA was recovered after elution with 75 l of EB elution buffer (Qiagen).

[0157] Recombinant Antibody Production

[0158] Human embryonic kidney (HEK) 293 (ATCC, No.CRL-1573) or 293T (ATCC, No. CRL-11268) cells were cultured in 150 mm plates (Falcon, Becton Dickinson) under standard conditions in Dulbecco's Modified Eagle's Medium (DMEM; GibcoBRL) supplemented with 10% heat-inactivated ultralow IgG fetal calf serum (FCS) (Invitrogen), 1 mM sodium pyruvate (GibcoBRL), 100 g/ml streptomycin, 100 U/ml penicillin G and 0.25 g amphotericin (all from GibcoBRL).

[0159] Transient transfections of exponentially growing HEK293 cells were performed by calciumphosphate precipitation at 80% cell confluency. Equal amounts (12.5-20 g each) of IgH and corresponding IgL chain expression vector DNA and 0.7 mM chloroquine (Sigma) were mixed in 1 ml sterile water and 2.5 M CaCl2 was added drop-wise to a concentration of 250 mM. An equal volume of 2HEPES-buffered saline (50 mM HEPES, 10 mM KCl, 12 mM Dextrose, 280 mM NaCl, 1.5 mM Na2HPO4-7H2O, pH 7.05) was mixed with the calcium-DNA solution under slow vortexing and incubated at room temperature for 10 min to allow formation of precipitates. The precipitation mixture was distributed evenly to the culture dish. The cells were washed with 10 ml serum-free DMEM after 8-12 h and cultured for 6 d in 25 ml DMEM supplemented with 1% Nutridoma-SP (Roche) before supernatants were harvested and analyzed by enzyme-linked immunosorbent assay (ELISA) for recombinant antibody production.

[0160] Recombinant Antibody Purification

[0161] Cell debris was removed by centrifugation at 800g for 10 min and culture supernatants were stored at 4 C. with 0.05% sodium azide. Recombinant antibodies were purified with Protein G beads (GE Healthcare) according to the manufacturer's instructions. In brief, 25 ml cell culture supernatant was incubated with 25 l Protein G beads for at least 14 h at 4 C. under rotation. Supernatants were removed after centrifugation at 800g for 10 min and the beads were transferred to a chromatography spin column (BioRad) equilibrated with PBS. After two rounds of washing with 1 ml PBS, antibodies were eluted in 3-4 fractions (200 l each) with 0.1 M glycine (pH 3.0). Eluates were collected in tubes containing 20 l 1 M Tris (pH 8.0) with 0.5% sodium azide. Recombinant antibody concentrations were determined by ELISA, all steps were performed at ambient temperature.

[0162] 1.2. Validation of Antibody Binding to Human NMDAR1 Protein and Pathogenic Effects

[0163] Transfected HEK293 Cells (FIG. 5)

[0164] The cDNA of the human ionotropic glutamate N-methyl-D-aspartate 1 receptor (GRIN1) was kindly provided by Prof. Dr. Wanker (MDC, Berlin) and cloned into pBudCE4.1 (Life Technologies). NR1 DNA (1 g) was mixed with 3 g PEI and 100 l 150 mM NaCl, vortexed and incubated for 10 min, and HEK293 cells were transiently transfected. Two days later, HEK293 cells on cover slips were fixed with methanol at 20 C. for 4 min. In addition, HEK293 cells transfected with a different NMDAR clone, leucine-rich glioma-inactivated 1 (LGI1), contactinassociated protein 2 (Caspr2), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, and gamma-aminobutyric acid b (GABAb) receptor were used (Autoimmune-Enzephalitis-Mosaik 1, Euroimmun, Lbeck, Germany). For staining with monoclonal antibodies and control antibodies, cells were washed in PBS, preincubated with 5% normal goat serum containing 2% bovine serum albumin and 0.1% Triton X-100, and incubated with antibodies starting at a 1:2 dilution of the cell culture supernatant overnight at 4 C. Secondary fluorescently labeled anti-human IgG antibodies were used for visualization. Sections were washed in PBS and coverslips mounted with Immu-Mount (ThermoScientific). Double-labeling of transfected cells was performed using commercial antibodies: monoclonal mouse and polyclonal rabbit anti-NR1 (1:100, Synaptic Systems) (FIG. 5) and reactive clones were identified.

[0165] The following reactive clones were identified (HC=heavy chain; LC=light chain; bold=antigen binding regions (CDR1-3) of the respective chains): Seq.-ID 1-14.

[0166] Brain Sections (FIG. 6)

[0167] Paraformaldehyde-fixed mouse and rat brain sections were used. Tissue was permeabilized in 0.1% Triton X-100 in PBS for 20 min and blocked in 10% normal goat serum for 30 min. Culture supernatants of transfected HEK293 cells containing monoclonal human recombinant or control antibodies were diluted 1:2 to 1:200 and sections incubated overnight at 4 C. Secondary fluorescently labeled anti-human IgG antibodies were used for visualization. Sections were washed in PBS and coverslips mounted with Immu-Mount (ThermoScientific).

[0168] Staining pattern of NR1-reactive clones was identical to the known anatomical distribution of NMDAR in the mouse hippocampus (FIG. 6).

[0169] Down-Regulation of NMDAR-Positive Synaptic Clusters

[0170] Primary hippocampal neurons were cultured after dissection from mouse brains. Hippocampi at embryonic day 16 were dissociated in MEM supplemented with 10% fetal calf serum, 100IE insulin/l, 0.5 mM glutamine, 100 U/ml penicillin/streptomycin, 44 mM glucose and 10 mM HEPES. Following centrifugation, cells were resuspended in serum-free neurobasal medium supplemented with B27, 0.5 mM glutamine, 100 U/ml penicillin/streptomycin and 25 M glutamate and 810.sup.4 cells/well plated on cover slips precoated with poly-L-lysine/collagen (all ingredients from Gibco/BRL). Cells were used for immunocytochemistry at day 14 in vitro to allow for full maturation of functional synapses.

[0171] For quantification of NMDAR-positive synaptic clusters, primary neurons were treated for 18 hours with monoclonal human recombinant anti-NMDAR1 antibody or control antibody (FIG. 7). After incubation with pathogenic and control antibodies, the cells were fixed and stained for the non-internalized fraction of NMDAR1s using commercial NMDAR antibodies (Synaptic Systems). Clusters were determined using 40 images of 100 m proximal dendrite length at 40 magnification per condition in each individual experiment. Images were converted to greyscale. Color-inverted and thresholded images were analyzed by Scion Image software (Scion, now http://en.bio-soft.net/) according to intensity and size criteria. For accurate comparison between treatment groups regarding areas of similar cell numbers, cell counts were performed. Down-regulation of NMDAR clusters with the monoclonal human NMDAR antibody (FIG. 7) is comparable to the data in the literature using whole CSF of NMDAR encephalitis patients.

[0172] Epitope Analysis

[0173] Point mutation N368Q was introduced into the NR1 construct using the Stratagene QuikChange Mutagenesis kit according to manufacturer's instructions and the mutant transiently transfected into HEK293 cells as described previously (Doss et al, 2014). Staining of HEK293 cells expressing natural and mutated NR1 construct was performed as described above. Binding to the mutant was eliminated for all monoclonal human NMDAR1 antibodies (FIG. 8).

Example 2

[0174] Generation of Antibodies Against Monoclonal Human Recombinant NMDAR1 Antibodies

[0175] A scFv library was constructed from patient PBMC as described in Frenzel et al. 2014. Methods Mol Biol. 1060:215-43. Panning was performed over three rounds on immobilized human monoclonal anti-NMDAR1-antibody and screening was performed by ELISA on immobilized antigen and myc-Tag-detection as described in Hust et al. 2014 Methods Mol Biol. 1101:305-20.

Example 3

[0176] Generation of Aptamers Against Monoclonal Human Recombinant NMDAR1 Antibodies

[0177] The generation of aptamers is conducted according to Jones et al. 2006. Antimicrob. Agents Chemother. 50(9): 3019-3027. SELEX (Ellington and Szostak 1990. Nature. 346(6287):818-22; Tuerk and Gold 1990. Science. 249(4968):505-10) was used to select for aptamers that recognize human monoclonal anti-NMDAR1-antibodies attached to cyanogen bromide (CNBr)-activated Sepharose via an N-terminal linker OR via Protein-A-Sepharose. A DNA library with a diversity of 10.sup.14 comprising a 40-nt random region flanked by two primer binding sites was in vitro transcribed to yield the respective RNA library. RNA was incubated with the selection matrix and after removal of nonbinding sequences by washing with binding buffer, remaining species were eluted, reverse-transcribed, and used as input DNA for the next transcription and a new selection cycle. Binding species were enriched after six cycles of selection, reverse-transcribed, cloned, and sequenced. Monoclones exhibiting better binding properties in column assays to anti-NMDAR1-antibody-Sepharose than the enriched pool from cycle 6 were selected for affinity determination.

Example 4

[0178] Assay for Determining the Binding Affinity of the Antibodies and Aptamers Against Monoclonal Human Recombinant NMDAR1 Antibodies

[0179] The affinity of selected antibodies or aptamers was measured by surface plasmon resonance (SPR) analysis (Jones et al. 2006. Antimicrob. Agents Chemother. 50(9):3019-3027). In more detail, the Biacore X platform (GE Healthcare) was used to perform binding analysis of the selected aptamers. Therefore, monoclonal NMDAR antibodies are immobilized onto protein A sensor chip (GE Healthcare) utilizing amine coupling as described (Schtze T, et al. 2011, PLoS ONE 6(12): e29604.). Binding analysis is conducted at a flow rate of 30 l/min with binding buffer at 25 C. Prior to injection, synthetic oligonucleotides are denatured for 3 min at 94 C. and refolded in binding buffer. 30 l of the aptamer solution in a range from 0.1 to 2.0 M are injected into the flow cell. After each aptamer injection, the chip surface was regenerated by injection of 210 l 0.5 mM NaCl/0.5 mM MgCl.sub.2. Association and dissociation rates and constants of the aptamer-streptavidin complexes are determined using BIAevaluation software (Biacore).

Example 5

[0180] 5.1. Inhibition of Binding (Neutralization) of Monoclonal Human Recombinant NMDAR1 Antibodies to NMDAR1-Expressing HEK293 Cells [0181] HEK293 cells were transiently transfected with the cDNA of the human ionotropic glutamate N-methyl-D-aspartate 1 receptor (Gene ID: GRIN1) and grown on coverslips for immunocytochemistry as described above. Specific aptamers or Ig or non-Ig scaffold were pre-incubated with monoclonal anti-NMDAR1 antibodies for 30 min at room temperature at 2-20 fold molar excess of aptamer vs anti-NMDR1 antibody. Controls contained either monoclonal antibody, non-anti-NMDAR1 control antibodies or aptamer only. Cells were washed in PBS, preincubated with 5% normal goat serum containing 2% bovine serum albumin and 0.1% Triton X-100 and incubated with aptamer-antibody mixtures or control antibodies overnight at 4 C. Secondary fluorescently labeled anti-human IgG antibodies were used for visualization. Coverslips were washed in PBS and mounted with Immu-Mount (ThermoScientific). Neutralization of antibody binding is determined by reduction of fluorescence signal to baseline levels, i.e. fluorescence intensity of cells incubated with the non-NMDAR binding control antibody.

[0182] 5.2. Inhibition of Binding of Monoclonal Human Recombinant NMDAR1 Antibodies to NMDAR1-Expressing Brain Sections [0183] Paraformaldehyde-fixed mouse and rat brain sections were used. Tissue was permeabilized in 0.1% Triton X-100 in PBS for 20 min and blocked in 10% normal goat serum for 30 min. Specific aptamers or blocking Ig or non-Ig scaffold were pre-incubated with anti-NMDAR1 monoclonal antibodies for 30 min at room temperature at 2-20 fold molar excess of aptamer vs anti-NMDR1 antibody. Controls contained either monoclonal antibody, non-NMDAR control antibodies or aptamer only. Sections were incubated overnight at 4 C. Secondary fluorescently labeled anti-human IgG antibodies were used for visualization. Sections were washed in PBS and coverslips mounted with Immu-Mount (ThermoScientific). The staining pattern was compared to the known anatomical distribution of NMDAR in the mouse hippocampus. Neutralization of antibody binding is determined by reduction of fluorescence signal to baseline levels, i.e. fluorescence intensity of sections incubated with the non-NMDAR binding control antibody (FIG. 11). Pre-incubation of anti-NMDAR monoclonal antibody with aptamer markedly reduces the binding of the anti-NMDAR monoclonal antibody to the NMDAR within the dentate gyrus of the hippocampus.

[0184] 5.3. Inhibition of Auto-Antibody-Mediated Downregulation of NMDAR-Positive Postsynaptic Clusters [0185] Primary hippocampal neurons were cultured and prepared for immunocytochemistry as described above. Cells were used at day 14 in vitro to allow full maturation of functional synapses. NMDAR-positive synaptic clusters on primary neurons were quantified as before comparing the staining by monoclonal human recombinant anti-NMDAR1 antibody with the pre-incubated aptamer-antibody mixture (molar ratio 20:1).

[0186] 5.4. Inhibition of Binding of NMDAR1-Positive Patient Serum Antibodies to NMDAR1-Expressing HEK293 Cells [0187] HEK293 cells were transiently transfected with the cDNA of the human ionotropic glutamate N-methyl-D-aspartate 1 receptor (GeneID: GRIN1) and grown on coverslips for immunocytochemistry as described above. Specific aptamers were pre-incubated with patient serum for 30 min at room temperature. Controls contained either control antibodies or patient serum only. Cells were washed in PBS, preincubated with 5% normal goat serum containing 2% bovine serum albumin and 0.1% Triton X-100 and incubated with sample mixtures or controls overnight at 4 C. Secondary fluorescently labeled anti-human IgG or IgA antibodies were used for visualization. Coverslips were washed in PBS and coverslips mounted with Immu-Mount (ThermoScientific). The fluorescence intensity of cells was recorded. Inhibition was determined by significant signal reduction in samples containing aptamers compared to samples with patient serum only.

Example 6: Sequence Analysis of Patient-Derived NMDAR-Autoantibodies

[0188] Patient-derived NMDAR-autoantibodies were isolated as described in Example 1. Sequences were analyzed by IgBLAST comparison with GenBank (Ye J et al. 2013. Nucleic Acids Res. 41). The CDRs were identified, aligned and analysed for CDR lengths, properties of CDR residues and sequence homology (FIG. 9). CDR sequence alignment revealed functional homology human NMDAR antibodies derived from different patients.

Example 7: Identification of Unmutated Human NMDAR-Autoantibodies (Kreye et al. 2016, Brain)

[0189] For each IgG sequence the number of somatic hypermutations in the immunoglobulin gene was counted in comparison to the annotated germline sequences as well as the length of the complementarity determining regions (Kabat and Wu, 1991; Kabat et al. 1983). Unmutated human antibodies against the NMDAR were identified (FIG. 10). These antibodies comprise the so-called germ-line configuration (also called naturally occurring antibodies), i.e. they are continuously generated by the body, not only in patients but in everyone and will thus stochastically be present also in previously healthy persons. Due to the sequence code of naturally occurring NMDAR antibodies in the normal (healthy) genetic repertoire, the data suggest that there is an evolutionary restriction to a limited number of sequences which might be relevant to larger numbers of people.

FIGURE DESCRIPTION

[0190] FIG. 1a:

[0191] Illustration of antibody formatsFv and scFv-Variants

[0192] FIG. 1b:

[0193] Illustration of antibody formatsheterologous fusions and bifunctional antibodies

[0194] FIG. 1c:

[0195] Illustration of antibody formatsbivalent antibodies and bispecific antibodies

[0196] FIG. 2:

[0197] Several rounds of selection and amplification result in highly specific and affine aptamers

[0198] FIG. 3:

[0199] First monoclonal recombinant NMDAR1 autoantibody

[0200] Technical overview

[0201] FIG. 4:

[0202] First monoclonal recombinant NMDAR1 autoantibody

[0203] FACS Sort Strategy

[0204] FIG. 5:

[0205] First monoclonal recombinant NMDA1R autoantibody

[0206] Staining of NR1-transfected HEK293 cells (diagnostic routine assay) confirming NMDAR-specific binding. (A) hNR1=human monoclonal anti-NR1 antibody, (B) msNR1=commercial mouse anti-NR1 antibody, (C) merged image demonstrating complete staining overlap.

[0207] FIG. 6:

[0208] First monoclonal recombinant NMDAR autoantibody.

[0209] Specific staining of hippocampus on mouse brain section.

[0210] FIG. 7:

[0211] First monoclonal recombinant NMDAR autoantibody.

[0212] NMDAR cluster downregulation in hippocampal primary neurons.

[0213] FIG. 8:

[0214] Epitope analysis of monoclonal human NMDAR1 antibodies

[0215] HEK293 cells were transfected with wildtype NR1 or a construct with mutation N368Q. As exemplarily shown for clone 007-168, all human monoclonal NMDAR1 antibodies strongly recognized wildtype NR1 (A), but staining of the mutant was eliminated (B).

[0216] FIG. 9:

[0217] CDR sequence comparison of human monoclonal NMDAR1 antibodies

[0218] CDR sequence alignment of human NMDAR antibodies from different patients reveals functional homology. Sequence annotation: * identity in 6/6 sequences; +identity or functional similarity in 5/6 sequences (A). Cross sequence similarities. L CDR2 (right): only 3 residues short=low sterical freedom (in 6/6); dominated by acidic residues (marked in bold (D,E), in 5/6). H CDR1 (left): similar length=8 or 9 residues (in 6/6); dominant aromatic residues (marked in bold (F, Y, W; in 5/6); grouped according to homology (B). NMDAR antibodies derived from different patients show high degree of homology. Comparison of CDR sequences of 003-109 and 007-169. Identity in L CDR1; homology in L CDR2, L CDR3 and H CDR1; similar acidic character in H CDR2 and H CDR3 (C).

[0219] FIG. 10:

[0220] Number of somatic hypermutations in recombinant human monoclonal NMDAR antibodies. For each generated antibody from antibody-secreting cells in the cerebrospinal fluid of encephalitis patients, the number of somatic hypermutations (SHM) for the V gene segments in the Ig heavy (IGH) and also of the corresponding Ig kappa (IGK) or lambda (IGL) light chains are plotted. The NR1-reactive antibodies (dark dots) show an average of 4.9 SHM in the IGHV and 4.1 SHM in the IGKV/IGLV gene segment which is much less in other (non-NR1) antibodies. Importantly, some NR1 antibodies have not a single hypermutation, thus reflecting naturally occurring antibodies.

[0221] FIG. 11:

[0222] Aptamers reduce the binding of monoclonal human NMDAR antibodies to mouse brain sections.

[0223] Monoclonal NMDAR antibodies strongly bind to the NMDAR-expressing areas in the murine hippocampus (the asterisk marks the dentate gyrus of the hippocampus which shows highest NMDAR protein expression) (A). Preincubation of the same antibodies with the enriched aptamer pool resulted in a marked reduction of antibody binding to the mouse brain (B).