NMDA RECEPTOR CONSTRUCTS TO DETECT AND ISOLATE NMDAR AUTOANTIBODIES

Abstract

A soluble N-methyl-D-aspartate receptor (NMDAR) protein construct includes one or more NMDAR autoantibody epitopes. The construct includes an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment of the subunit and an ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment of these subunits. An in vitro method for the detection of NMDAR autoantibodies in a sample includes providing a sample suspected of including NMDAR autoantibodies, providing the NMDAR protein construct as a capture molecule, contacting the sample with the NMDAR protein construct, thereby binding NMDAR autoantibodies from the sample to the NMDAR protein construct, and determining the presence and optionally the amount of bound NMDAR autoantibodies. The method is applied for the diagnosis, prognosis, disease monitoring, patient stratification and/or therapy monitoring of a medical condition associated with autoantibodies against the NMDAR, preferably anti-NMDAR encephalitis.

Claims

1. An N-methyl-D-aspartate receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, wherein the construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof and an ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment thereof, wherein the protein construct lacks a NMDAR transmembrane domain.

2. (canceled)

3. The NMDAR protein construct according to claim 1, wherein the construct comprises a dimerization domain and/or a capture domain.

4. The NMDAR protein construct according to claim 3, wherein the dimerization domain is the capture domain, preferably formed by an antibody Fc-fragment.

5. The NMDAR protein construct according to claim 1, wherein the ECD of GluN1 or fragment thereof comprises or consists of the amino terminal domain (ATD) of GluN1 or a fragment thereof, and/or wherein the ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment thereof comprises or consists of the ATD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment thereof, respectively.

6. The NMDAR protein construct according to claim 1, wherein the ECD of GluN1 and the ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment thereof, are covalently linked, preferably as a fusion protein.

7. The NMDAR protein construct according to claim 1, wherein the construct is a protein dimer of non-covalently bound monomers, wherein the construct can be a homodimer or a heterodimer.

8. The NMDAR protein construct according to claim 7, wherein the construct is a heterodimer formed from the ECD of GluN1 or fragment thereof (as one monomer) and the ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment thereof (as one monomer).

9. An in vitro method for the detection of NMDAR autoantibodies in a sample, the method comprising, a. providing a sample suspected of comprising NMDAR autoantibodies, b. providing a NMDA protein construct according to claim 1 comprising a capture domain as a capture molecule, c. contacting said sample with said NMDAR protein construct, thereby binding NMDAR autoantibodies from said sample to said NMDAR protein construct, and d. determining the presence of bound NMDAR autoantibodies.

10. The method according to claim 9, wherein the NMDAR autoantibodies in said sample are present in solution or on a cell-membrane.

11. The method according to claim 9, wherein the method is carried out with multiple and different of the NMDAR protein constructs.

12. The method according to claim 9, wherein the method is applied for the diagnosis, prognosis, disease monitoring, patient stratification and/or therapy monitoring of a medical condition associated with autoantibodies against the NMDAR, preferably anti-NMDAR encephalitis, and the sample suspected of comprising NMDAR autoantibodies is a sample of a human subject exhibiting symptoms of having said medical disorder.

13. The method according to claim 9, wherein the method is applied for therapy guidance of a subject suspected of having and/or developing a medical condition associated with NMDAR autoantibodies, the method comprising selecting one or more corresponding NMDAR protein construct(s) for subsequent treatment of said subject.

14. A kit for the diagnosis of an autoimmune disease associated with NMDAR autoantibodies in a subject by detection of NMDAR autoantibodies, comprising: a. an NMDAR protein construct according to claim 1 or an NMDAR protein construct according to claim 1 immobilized on a solid surface, and a labelled secondary affinity reagent directed to human NMDAR autoantibodies and a detector for detecting the signal emitted from said label, or b. a labelled NMDAR protein construct according to claim 1.

15. A blood treatment device configured to remove NMDAR autoantibodies from the blood or blood plasma of a person in need thereof in an extracorporeal blood circuit, wherein the device comprises a matrix having one or more NMDAR protein constructs according to claim 1 immobilized thereon.

16. The method according to claim 11, further comprising additionally determining against which NMDAR protein construct of said multiple constructs the NMDAR autoantibodies bind.

17. The method according to claim 11, further comprising additionally determining against which NMDAR protein construct of said multiple constructs the NMDAR autoantibodies bind in the largest amounts and/or most efficiently bind.

18. The kit according to claim 14, wherein the disease associated with NMDAR autoantibodies is-NMDAR encephalitis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0204] FIG. 1: Scheme of soluble recombinant NMDA receptor Fc (srNR-Fc) antigens/fusion proteins. In the context of the invention, srNR-Fc antigen and srNR-Fc fusion protein are used interchangeably.

[0205] FIG. 2: Soluble recombinant NMDA receptor Fc fusion proteins are recognized by a recombinant human GluN1 autoantibody.

[0206] FIG. 3: Soluble recombinant NMDA receptor Fc fusion proteins detect NMDA receptor autoantibodies in patients' sera.

[0207] FIG. 4: Autoantibody detection by soluble recombinant NMDA receptor Fc fusion proteins in sera with known anti-NMDA receptor titers.

[0208] FIG. 5: ELISA screen for LGI1 antibodies using a soluble recombinant NMDA receptor Fc fusion protein (fusion protein #1 as disclosed herein) as a control.

[0209] FIG. 6: ELISA quantification of recombinant human NR1(GluN1) AB in mouse brain extracts using a soluble recombinant NMDA receptor Fc fusion protein according to fusion protein #1 disclosed herein.

[0210] FIG. 7: Soluble NMDA receptor Fc protein constructs of the invention detect heterodimer-selective recombinant human NMDA receptor autoantibodies.

[0211] FIG. 8: Soluble NMDA receptor Fc protein constructs of the invention detect subtype-selective recombinant human NMDA receptor autoantibodies.

[0212] FIG. 9: Soluble NMDA receptor Fc protein constructs of the invention reveal the subtype selectivity of recombinant human NMDA receptor autoantibodies.

[0213] FIG. 10: Detection of recombinant human NMDA receptor autoantibodies by three soluble NMDA receptor Fc protein constructs of the invention.

DETAILED DESCRIPTION OF THE FIGURES

[0214] FIG. 1. A. Simplified representation of NMDA receptor subunits encompassing amino terminal domain (ATD), ligand-binding domain (LBD), plasma membrane (PM)-spanning/associated segments (M1-4) and intracellular carboxyterminal domain (CTD). For details please see Paoletti et al. (2013) Nature Rev Neurosci 14, 383ff. B. Extracellular domains of NMDA receptor subunits depicted in A fused to rabbit Fc (rbFc, black triangle represents a single polypeptide containing CH2 and CH3) yield soluble antigens. The black line represents a linker between domains derived from different subunits (for details see Table 3). C. The soluble antigens are expected to form homo- or heteromeric dimers through rbFc upon (co)expression (a selection of the possible combination is shown).

[0215] FIG. 2. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate through an anti-rabbit IgG antibody. Single recombinant (rec) human (hu) autoantibodies to LGI1 or GluN1 (Kreye et al., 2016) derived from patients' CSF cells were applied at 1 μg/ml and detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody; captured Fc fusion proteins were detected directly using an HRP-conjugated anti-rabbit (rb) IgG antibody at 0.016 μg/ml. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0216] FIG. 3. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate through an anti-rabbit Fc antibody. Sera derived from seven NMDAR encephalitis patients (S3-9) and a control serum (ctrl S) were applied at a 1:200 dilution and human IgG detected using Biotin-conjugated anti-human IgG and HRP-conjugated streptavidin. Panels A to C represent separate experiments. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal caused by the combination of Biotin-conjugated anti-human IgG and HRP-conjugated streptavidin alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0217] FIG. 4. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins were captured on a 96-well plate through an anti-rabbit Fc antibody. Sera derived from five NMDAR encephalitis patients (S10-14) and three control sera (S15, S18, S19) were applied at a 1:100 dilution and human IgG detected using Biotin-conjugated anti-human IgG and HRP-conjugated streptavidin. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal caused by the combination of Biotin-conjugated anti-human IgG and HRP-conjugated streptavidin alone. NMDAR antibody titers of the sera determined by the Euroimmun cell-based assay are given below for comparison. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0218] FIG. 5. To screen for LGI1 reactivity, cell culture supernatants of HEK293T cells expressing CSF cell-derived antibody cDNAs were applied to LGI1-Fc and to NMDA receptor subunit GluN1-ATD-Fc captured on an ELISA plate. Example assay shows results for 13 supernatants as well as controls including CSF samples (diluted 1:5), human recombinant anti-GluN1, mouse anti-LGI1 and secondary antibodies to human, mouse and rabbit IgG alone. Signals are shown as mean±SD of two wells.

[0219] FIG. 6. NR1 (GluN1) specific human IgG was found in neonatal mouse whole brain extracts via ELISA only following prenatal NR1 antibody injections with increasing concentrations between PO and P7. ELISA quantification of NR1-specific human AB in mouse brain extracts revealed increasing levels of brain-bound IgG from PO (mean=10.7 ng) to P7 (mean=37.4 ng) in the NR1 group.

[0220] FIG. 7. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate through an anti-rabbit IgG antibody. Single recombinant human NMDA receptor autoantibodies (anti-NR-Ab1 and 2) derived from patients' CSF cells or a control antibody (mGO53) were applied at 4.0 μg/ml (anti-NR-Ab1), 3.1 μg/ml (anti-NR-Ab2) and 5.9 μg/ml (mGO53) and were detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody; captured Fc fusion proteins were detected directly using an HRP-conjugated anti-rabbit (rb) IgG antibody at 0.016 μg/ml. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. A 490 nm values are shown for anti-rabbit IgG. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0221] FIG. 8. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate through an anti-rabbit IgG antibody. Single recombinant human NMDA receptor autoantibodies derived from patients' CSF cells (Kreye et al., 2016 or unpublished) or a recombinant human control antibody (control Ab1) were applied at 1 μg/ml and were detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody; captured Fc fusion proteins were detected directly using an HRP-conjugated anti-rabbit (rb) IgG antibody at 0.016 μg/ml. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0222] FIG. 9. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate through an anti-rabbit IgG antibody. Single recombinant human NMDA receptor autoantibodies derived from patients' CSF cells (Kreye et al., 2016 or unpublished) or a control antibody (mGO53) were applied at 0.09 μg/ml (003-102) or 1.8 μg/ml (anti-NR-Ab1, mGO53) and were detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody; captured Fc fusion proteins were detected directly using an HRP-conjugated anti-rabbit (rb) IgG antibody at 0.016 μg/ml. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. A 490 nm values are shown for 003-102 and for anti-rabbit IgG. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0223] FIG. 10. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate through an anti-rabbit IgG antibody. Single recombinant human NMDA receptor autoantibodies derived from patients' CSF cells (Kreye et al., 2016 or unpublished) were applied at 0.01 μg/ml (003-102), 1 μg/ml (008-218, anti-NR-Ab1) or 10 μg/ml (all others) and were detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody; captured Fc fusion proteins were detected directly using an HRP-conjugated anti-rabbit (rb) IgG antibody at 0.016 μg/ml. ELISA signals are shown as mean±SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0224] Concerning the FIGS. 2-10, in each of the figures the order of the tested constructs in the legend from top to bottom corresponds to the order of the bars from left to right for the respective condition.

Examples

[0225] The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0226] Technical Question

[0227] Is it possible to generate recombinant soluble fusion proteins for labelling, detection and isolation of NMDA receptor autoantibodies against NMDA receptors of different subunit compositions within the serum and CSF of patients?

Solution

[0228] The amino terminal domain (ATD) of the NMDA receptor subunit GluN1, with or without additional extracellular domains of GluN1, alone or combined with extracellular domains of the NMDA receptor subunits GluN2A or GluN2B were fused to the constant region of rabbit IgG1 heavy chain (rbFc).

[0229] Fc mediated dimerization of expressed proteins may lead to epitopes highly similar to native NMDA receptors and may lend unprecedented stability to the fusion proteins. These soluble recombinant NMDA receptor Fc (srNR-Fc) fusion proteins/antigens are able to detect NMDA receptor autoantibodies against different NMDA receptor subunit compositions present in the serum of NMDAR encephalitis patients and therefore represent the core subject matter of this invention.

[0230] The ELISA method used to detect NMDA receptor autoantibodies with srNR-Fc fusion proteins/antigens may be used as a companion diagnostic.

Detailed Examples

Example 1: Exemplary Protein Constructs of the Invention Generated for Experiments

[0231] We have generated constructs encompassing extracellular parts of GluN1 and GluN2 subunits (FIGS. 1A and 1B, Table 1), expressed them in HEK293 cells and isolated cell culture supernatants containing the secreted Fc fusion proteins three days later.

[0232] Constructs #1, 2, 3, 5, 6 and 9 (Table 3) are Fc fusion proteins of either GluN1 or GluN2 domains, whereas in constructs #4, 7 and 8 domains of both GluN1 and GluN2B, separated by an artificial linker, are fused to Fc in a single molecule. The Fc domain will likely lead to dimerization of all the fusion proteins, leading to GluN1/GluN2 heterodimers upon co-expression of construct #1 or 2 with #5, 6, 9 or 3, respectively, or dimers of GluN1/GluN2 heterodimers upon expression of constructs #4, 7 and/or 8 (FIG. 1C).

TABLE-US-00003 TABLE 3 Soluble recombinant NMDA receptor rbFc fusion proteins that are comprised by and/or represent NMDAR protein constructs of the invention. NMDA receptor part: amino acids in GenBank entry Fusion Protein No of amino acids in NMDA receptor # Designation Composition part/whole protein 1 N1-ATD-Fc hsGluN1-ATD-rbFc 1-400 (NM_007327) 400/631 2 N1ecd-Fc hsGluN1-ATD-S1-GT- 1-544-GT-663-800 (NM_007327) S2-rbFc 684/915 3 N2Becd-Fc hsGluN2B-ATD-S1-GT- 1-540-GT-662-803 (NM_000834) S2-rbFc 684/915 4 N1ecd-N2Becd- hsGluN1-ATD-S1-GT- 1-544-GT-663-800 (NM_007327)-GST- Fc S2-linker-hsGluN2B- 3xGGGGS-GAA-SR- ATD-S1-GT-S2-rbFc 29-540-GT-662-803 (NM_000834) 1363/1594 5 N2A-ATD-Fc hsGluN2A-ATD-rbFc 1-408 (NM_001134407) 408/639 6 N2B-ATD-Fc hsGluN2B-ATD-rbFc 1-408 (NM_000834) 408/639a 7 N1-ATD-N2A- hsGluN1-ATD-linker- 1-400(NM_007327)-GST-3xGGGGS-GAA-SR- ATD-Fc hsGluN2A-ATD-rbFc 28-408 (NM_001134407) 804/1035 8 N1-ATD-N2B- hsGluN1-ATD-linker- 1-400(NM_007327)-GST-3xGGGGS-GAA-SR- ATD-Fc hsGluN2B-ATD-rbFc 29-408(NM_000834) 803/1034 9 N2C-ATD-Fc hsGluN2C-ATD-rbFc 1-405 (NM_000835) 405/636a hs, human. rb, rabbit. Ecd, extracellular domain. ATD, amino terminal domain. No, number. Fc, constant region of rabbit IgG1 heavy chain.

[0233] We established an ELISA to test the ability of srNR-Fc proteins to detect NMDA receptor antibodies in the serum of patients. Briefly, srNR-Fc proteins in cell culture supernatants were captured on 96-well plates via anti-rabbit Fc or anti-rabbit IgG antibodies. NMDAR encephalitis patient sera or human monoclonal antibodies were applied and bound antibody detected using either Biotin-conjugated anti-human IgG and horseradish peroxidase (HRP)-conjugated streptavidin or HRP-conjugated anti-human IgG antibody, and the HRP substrate ultraTMB.

Example 2: Soluble NMDA Receptor rbFc Fusion Proteins are Recognized by a Recombinant Human GluN1 Autoantibody

[0234] FIG. 2 shows that all srNR-Fc antigens tested, but not a control antigen (Progranulin-Fc) were recognized by a recombinant human anti-NMDA receptor antibody. In contrast, none of the antigens was recognized by a recombinant human anti-LGI1 antibody. An anti-rabbit IgG antibody detecting the rbFc portion of the fusion proteins was used to confirm that all rbFc fusion proteins were successfully expressed and immobilized on the ELISA plate.

Example 3: Soluble NMDA Receptor rbFc Fusion Proteins Detect NMDA Receptor Autoantibodies in Patients' Sera

[0235] FIG. 3 summarizes results of three ELISA experiments with a set of human sera from NMDAR encephalitis patients on the different srNR-Fc antigens. The data reveals that GluN1-ATD-Fc as well as antigens containing additional extracellular regions of GluN1 or GluN2 subunits yielded signals as in most of the NMDAR encephalitis patient sera, compared to Progranulin as a control. Three of the sera strongly reacted with the antigens; the other four sera reacted only with selected srNR-Fc antigens and yielded lower signals. Sera were diluted 1:200 suggesting that the srNR-Fc antigens allow high sensitivity NMDA receptor autoantibody detection. Addition of regions of GluN2 subunits improved the sensitivity of the detection. A subset of NMDAR autoantibodies may recognize GluN1 only in the presence of an assembled GluN2 domain. The antigen yielding the highest signal differed from serum to serum. For example, antigen containing N1ecd-N2Becd was superior in detecting antibodies in serum 5, whereas antigens containing GluN1-ATD and GluN2B-ATD worked better in the detection of antibodies in serum 7.

Example 4: Autoantibody Detection by Soluble NMDA Receptor rbFc Fusion Proteins in Sera with Known Anti-NMDA Receptor Titers

[0236] To test how the ELISA based on srNR-Fc proteins compares to the clinical standard assay, we went on to measure sera with a known titer from the Euroimmun CBA (FIG. 4). All sera that had scored positive at Euroimmun (S10-14) yielded a positive signal with at least one of the antigens tested as compared to Progranulin and GluN2B-ATD as controls, while the sera that scored negative in the Euroimmun CBA (S15, S18, S19) did not. These data indicate that the srNR-Fcs containing GluN1-ATD specifically detect NMDAR autoantibodies in sera.

[0237] We used two srNR-Fc combinations expressing the ATDs of GluN1 and GluN2B in this assay (N1-ATD-Fc+N2B-ATD-Fc and N1-ATD-N2B-ATD-Fc). They contain the same amino acids of the NMDAR (Table 1), but in one case the antigen is reconstituted from two separate proteins, while the other construct contains the ATDs connected by an artificial linker as a single protein. These two antigens gave comparable signals with sera S10-S12, but differed considerably in sera S13 and S14. Antigen N1-ATD-Fc+N2B-ATD-Fc yielded small, comparable signals in S13 and S14. In contrast, N1-ATD-N2B-ATD-Fc did not detect any NMDAR antibody signal in S13 and a strong signal in S14. The molecular make-up of N1-ATD-N2B-ATD-Fc may have prevented access of the NMDAR autoantibodies present in S13 to their epitope. This finding emphasizes that several srNR-Fc combinations should be tested to detect as many antibodies as possible.

Example 5: Soluble NMDA Receptor rbFc Fusion Proteins Detect Subtype-Selective Recombinant Human NMDA Receptor Autoantibodies

[0238] Some autoantibodies were detected by soluble NMDA receptor Fc antigens encompassing the extracellular domains of two different NMDA receptor subunits, but not by soluble NMDA receptor Fc antigens containing the extracellular domains of a single NMDA receptor subunit (FIG. 7). These NMDA receptor antibodies are not detected by assays based on GluN1-expressing cells.

[0239] The soluble NMDA receptor Fc antigens were used to determine if a specific subunit combination is targeted by recombinant human NMDA receptor autoantibodies. NMDA receptor autoantibody 008-218 was detected by soluble NMDA receptor Fc antigens either containing the ATDs of GluN1 and GluN2A or containing the ATDs of GluN1 and GluN2B with a comparable efficiency. However, anti-NR-Ab1 was detected by soluble NMDA receptor Fc antigens containing the ATDs of GluN1 and GluN2B, but not by soluble NMDA receptor Fc antigens containing the ATDs of GluN1 and GluN2A (FIG. 8). Furthermore, this antibody was not detected by an additional soluble NMDA receptor Fc antigen containing the ATDs of GluN1 and GluN2C (FIG. 9). The soluble NMDA receptor Fc antigens therefore classify anti-NR-Ab1 as a GluN1/GluN2B-subtype-selective antibody.

[0240] The soluble recombinant NMDA receptor Fc antigen N1-ATD-N2B-ATD-Fc yielded higher signals than the assembled antigen N1-ATD-Fc+N2B-ATD-Fc with the GluN1/GluN2B-subtype-selective antibody anti-NR-Ab1 (FIG. 8), suggesting that the specific molecular make-up of the Fc fusion protein N1-ATD-N2B-ATD-Fc, with the ATDs of GluN1 and GluN2B being part of a single protein, provides an advantage in detecting subtype-selective antibodies.

Example 6: Detection of Recombinant Human NMDA Receptor Autoantibodies by Three Soluble NMDA Receptor rbFc Fusion Proteins

[0241] In a test of three soluble recombinant NMDA receptor Fc antigens, N1-ATD-N2B-ATD-Fc yielded the highest signals with several of the examined human recombinant NMDA receptor autoantibodies, while N1-ATD-Fc+N2B-ATD-Fc yielded a comparable or higher signal for others (FIG. 10). These data indicate a differential antibody-specific sensitivity of the soluble recombinant NMDA receptor Fc antigens and complement the findings in sera described in Example 4.

[0242] Discussion of the Examples

[0243] The results provided here serve as a proof of concept. We conclude (1) that soluble fusion proteins containing the amino terminal domain of GluN1 and rabbit Fc heterologously expressed in and secreted from HEK293 cells are able to bind NMDA receptor autoantibodies in patients' serum and (2) that efficient detection of NMDA receptor autoantibodies by soluble antigens benefits from the incorporation of extracellular domains of GluN2. Furthermore, use of the different srNR-Fc antigens may allow classifying patients' anti-NMDA receptor immune response.

[0244] Detection of autoreactivity against select NMDA receptor subtypes may enable a differential diagnosis in anti-NMDA receptor encephalitis and in other medical conditions associated with antibodies to the NMDA receptor.

Further Examples of Experimental Applications of Constructs of the Invention

[0245] ELISA Screen for Recombinant LGI1 Antibodies Using an NMDA Receptor Fusion Protein as a Control.

[0246] For generating the mammalian expression constructs used in this experiment the cDNAs for amino acids 1-558 of human LGI1 (NM_005097.3) and for amino acids 1-400 of human GluN1 (NM_007327) were inserted into pFuse-rIgG-Fc1 (InvivoGen). The resulting plasmids encode hsLGI1 or the amino terminal domain (ATD) of hsGluN1 fused to the Fc region of rabbit IgG (amino acids SKP-PGK) linked by amino acids GSSTMVRS. The chimeric constructs LRR1-EPTP2 and LRR2-EPTP1 encode rabbit Fc fusions of amino acids 1-223 of LGI1 and amino acids 218-545 of LGI2 or amino acids 1-217 of LGI2 and amino acids 224-557 of LGI1, respectively.

[0247] Antibody binding to LGI1-Fc and to NMDA receptor subunit GluN1-ATD-Fc was compared in an ELISA. 96-well high-binding microplates (Greiner #655061) coated with donkey anti-rabbit IgG (10 μg/mL, Dianova, #711-005-152) were blocked and incubated with cell culture supernatants of HEK293 cells that expressed Fc fusion proteins. Cell culture supernatants containing monoclonal antibodies, CSF samples or purified antibodies and horseradish peroxidase (HRP)-conjugated donkey-anti-human IgG (1:5,000, Dianova, #709-035-149) were sequentially applied. After thorough washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The presence of immobilized antigens was confirmed by incubation with HRP-conjugated F(ab′)2 donkey-anti-rabbit IgG (1:50,000, Dianova, #711-036-152). Human recombinant anti-GluN1 antibody 003-102 (Kreye J, Wenke N K, Chayka M, et al. Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis. Brain 2016; 139:2641-2652) was used at 10 ng/ml. The results are displayed in FIG. 5.

[0248] ELISA Quantification of Recombinant Human NR1(GluN1) AB in Mouse Brain Extracts Using an NMDA Receptor Fusion Protein.

[0249] Concentration of recombinant human NR1 AB #003-102 in brain extracts was determined in 96-well plates coated overnight at 4° C. with donkey-anti-rabbit IgG (20 μg/mL, Dianova, #711-005-152). After blocking with 2% BSA in PBS/0.05% Tween-20 (PBS/T) at RT, cell culture supernatants of HEK293 cells that expressed the amino terminal domain (amino acids 1-400) of human NR1 (GluN1) fused to rabbit Fc were applied. Mouse brain extracts were diluted 1:25/1:100 in 0.4% BSA-PBS/T and added in duplicates. Plates were washed with PBS/T and incubated with horseradish peroxidase (HRP)-conjugated donkey-anti-human IgG (1:5,000, Dianova, #709-035-149). After washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The concentrations of #003-102 in the extracts were deduced from a calibration curve generated with purified #003-102. The results are displayed in FIG. 6.

REFERENCES

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