EP4028769A1 - Détection améliorée des auto-anticorps du récepteur nmda - Google Patents

Détection améliorée des auto-anticorps du récepteur nmda

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Publication number
EP4028769A1
EP4028769A1 EP20767572.9A EP20767572A EP4028769A1 EP 4028769 A1 EP4028769 A1 EP 4028769A1 EP 20767572 A EP20767572 A EP 20767572A EP 4028769 A1 EP4028769 A1 EP 4028769A1
Authority
EP
European Patent Office
Prior art keywords
cell
variant
nmda receptor
human
carrier
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20767572.9A
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German (de)
English (en)
Inventor
Christian Probst
Swantje Mindorf
Ínga-Madeleine DETTMANN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Euroimmun Medizinische Labordiagnostika AG
Original Assignee
Euroimmun Medizinische Labordiagnostika AG
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Publication of EP4028769A1 publication Critical patent/EP4028769A1/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2857—Seizure disorders; Epilepsy

Definitions

  • the present invention relates to a carrier for microscopic immunofluorescence analysis coated with a eukaryotic cell overexpressing a polypeptide comprising human NMDA receptor or a variant thereof, a carrier comprising the cell and a mock-transfected cell, wherein the cell and the mock-transfected cell are spatially separated, a kit comprising the cell or the carrier and a use of the cell or the carrier or a polypeptide comprising human NMDA receptor or a variant thereof for producing an artefact-free signal for the detection of an autoantibody to human NMDA receptor.
  • NBDAR NMDA type glutamate receptor
  • NMDARs belong to the ionotropic glutamate receptors and were named according to their ability to be activated by the synthetic amino acid N-methyl-D-aspartate (NMDA). They are localized in the post-synaptic membrane and form cation canals with major significance for synaptic transmission and plasticity.
  • the receptors consist of two subunits, NR1 and NR2. Their activity is regulated by the binding of ligands, such as the neurotransmitter glutamate.
  • ligands such as the neurotransmitter glutamate.
  • autoimmune encephalitis are autoantibodies directed against an extracellular epitope of the NR1 subunit. These can be determined by immunohistochemical detection procedures or recombinant assays.
  • NMDA receptor autoimmune encephalitis a virtually stereotypical clinical course occurring in phases is typical.
  • a flu-like prodromal phase subfebrile temperature, headache, and fatigue
  • a psychotic stage with severe behavioral and personality changes, delusions, disturbed thoughts and hallucinations.
  • a drug-induced psychosis is initially diagnosed.
  • phase consciousness disorders hypoventilation, epileptic attacks, autonomous instability and l dyskinesia develop. Due to the severity of this disease including symptoms such as coma, status epilepticus, etc. many affected individuals are treated in intensive care for long periods of time.
  • ovarian tumors In the majority of young female patients ovarian tumors (teratoma) are found, which amongst other things contain nerve cells. These cases involve a paraneoplastic syndrome (PNS) in NMDA receptor autoimmune encephalitis. The probability of an associated tumor disease is, on average, around 60%, although this is dependent on age and gender. NMDA receptor autoimmune encephalitis is increasingly diagnosed not only in young women, but also in older patients, in women without teratoma, in men (some with teratoma of the testis) and in children.
  • PPS paraneoplastic syndrome
  • Prognosis for patients is improved with appropriate immunomodulatory therapy, and, in PNS, tumor detection and resection as early as possible. In around 75% of cases a substantial regression of symptoms can be achieved. However, 25% of patients die or suffer from severe neurological deficits. Survivors have memory loss for the duration of the illness, and there is a risk of relapses of the encephalitis syndrome, the latter in particular when the tumor is removed too late or not at all or if no tumor could be found.
  • NMDA receptor autoimmune encephalitis Diagnosis of NMDA receptor autoimmune encephalitis is based on a combination of the characteristic clinical picture, together with the supporting results from brain MRT, EEG and CSF analysis if necessary, and the detection of anti-glutamate receptor NMDA type antibodies. Infectious encephalitides, especially HSV and other autoimmune aetiologies (limbic encephalitis with autoantibodies against Hu, Ma2, CV2 and amphiphysin) must be excluded by differential diagnostics. In general, antibodies against NMDA receptors should be determined in all patients with encephalitis where no pathogen has been detected and in suspected cases of limbic encephalitis. When a positive serological result is obtained a comprehensive teratoma investigation should be undertaken.
  • IFT Indirect immunofluorescence
  • a kit comprising transfected cells for in vitro diagnostic use is commercially available (EUROIMMUN Medizinische Labordiagnostika AG, product number FA 112d-1003-51). It may be used for the high-throughput-screening of hundreds of patient samples in an automated manner under high-throughput-conditions. An excellent sensitivity of 98.1% and a specificity of 100% have been reported for this assay format.
  • discrepancies in the results between and results obtained using standardized immunofluorescence kit products and other assay formats have been observed. While it is unclear whether such discrepancies are due to flaws associated with these commercial kits or any reference assays, there remains the need for continuous optimization.
  • Dalmau et al. disclose that paraneoplastic NMDA receptor autoimmune encephalitis associated with ovarian teratoma is associated with autoantibodies to the NR1/NR2 heteromer of the receptor.
  • HEK293 cells transfected with plasmids containing rodent NR1, NR2A or NR2B subunits of the NMDA receptor were used.
  • McCracken et al. (2017) claim that standard clinical laboratory kits are specific but in rare cases yield inconclusive results or turn out insensitive or false negative results. (McCracken, L., Zhang, J., Greene, M., Crivaro, A., Gonzales, J., Kamoun, M., Lancaster, E. (2017) Improving the antibody-based evaluation of autoimmune encephalitis, Neurology.org/nn).
  • Probst et al. (2014) discuss technical challenges for developing and improving standardized serological test systems for the detection of autoantibodies, including NMDA receptor autoantibodies (Probst, C., Saschenbrecker, S., Stoecker, W., Komorowski, L. (2014) Anti- neural autoantibodies: Current diagnostic challenges. Multiple Sclerosis and Related Disorders. 3, 303-320).
  • US7,972,796 discloses methods of diagnosing or determining a cause of an autoimmune encephalitis comprising the step of testing a biological sample for an antibody to NR1 subunit of the NMDA receptor.
  • a problem underlying the present invention is to provide a new test and related creations, methods and uses for the diagnosis or aiding in the diagnosis of neurological autoimmune diseases, in particular NMDA receptor autoimmune encephalitis, at the utmost degree of diagnostic reliability, with special emphasis on sensitivity and for specificity.
  • the problem is solved by a carrier for microscopic immunofluorescence analysis coated with a eukaryotic, preferably mammalian cell overexpressing a polypeptide comprising human NMDA receptor or a variant thereof.
  • the problem underlying the present invention is solved by a carrier comprising the cell according to the present invention and preferably a mock-transfected cell, wherein the cell and the mock-transfected cell are spatially separated, wherein the carrier is preferably a slide for microscopic immunofluorescence analysis.
  • the cell is transiently transfected with human NMDA receptor or a variant thereof.
  • the cell is permanently expressing human NMDA receptor or a variant thereof.
  • the cell is a fixed cell.
  • the cell is fixed using formalin or acetone.
  • the cell is bound to an autoantibody to human NMDA receptor which is preferably bound to a secondary antibody.
  • the carrier further comprises one or more additional cells, preferably all from the group comprising a cell overexpressing GABA B or a variant thereof, a cell overexpressing LGI1 or a variant thereof, a cell overexpressing AMPAR1/R2 or a variant thereof, a cell overexpressing CASPR2 or a variant thereof, a cell overexpressing DPPX or a variant thereof, a cell overexpressing Hu or a variant thereof, a cell overexpressing Ma2 or a variant thereof, a cell overexpressing CV2 or a variant thereof and a cell overexpressing amphiphysin or a variant thereof, which are spatially separated from the cell overexpressing human NMDA receptor and, if present, the mock-transfected cell.
  • additional cells preferably all from the group comprising a cell overexpressing GABA B or a variant thereof, a cell overexpressing LGI1 or a variant thereof, a cell overexpressing AMPAR1/R2 or a variant thereof, a cell overexpressing CASPR2 or a variant thereof, a cell overexpressing DPP
  • the problem underlying the present invention is solved by a kit comprising the cell or the carrier according to the present invention, further comprising one or more reagents from the group comprising a secondary antibody, preferably labeled with a detectable label, a wash solution, a positive control, a negative control, a detergent, a cover glass, a mounting medium and a physiological salt solution, preferably PBS.
  • the problem is solved by a use of a vector comprising a nucleic acid encoding for a polypeptide comprising human NMDA receptor or a variant thereof or the cell according to the present invention for the manufacture of a kit or carrier or composition for the diagnosis of NMDA receptor autoimmune encephalitis, preferably the kit according to the present invention.
  • the problem is solved by a method comprising the steps a) contacting the carrier according to the present invention with a sample, b) washing the cell and c) detecting an autoantibody to human NMDA receptor bound to the carrier in step a), optionally an autoantibody to a neurological autoantigen overexpressed by another cell if a carrier comprising a second cell is used.
  • the autoantibody is detected in step c) using a secondary antibody labeled with a fluorescent dye.
  • the problem is solved by a use of a polypeptide comprising human NMDA receptor or a variant thereof or the cell or the carrier for producing artefact-free signals for the detection of an autoantibody to NMDA receptor.
  • the problem is solved by a use of a polypeptide comprising human NMDA receptor or a variant thereof or the cell or the carrier for the high specificity diagnosis of NMDA receptor autoimmune encephalitis.
  • the variant of the NMDA receptor is the NR1 subunit or a variant thereof.
  • the present invention is based on the inventors’ surprising finding that overexpression of rodent NMDA receptor or subunits thereof or variants thereof in eukaryotic cells is associated with the emergence of artefacts, in particular long protrusions of cells and unusually symmetrical spherical artefacts all of which stain positive when contacted with secondary antibodies with a label. These artefacts obscure images of positive stained cells or may be confused with cells specifically recognized by NMDA receptor autoantibodies, thus confusing untrained medical staff and leading to false positive or false negative diagnoses.
  • the present invention is based on the inventors’ surprising finding that artefact-free signals, preferably images for immunofluorescence analysis may be obtained if cells overexpressing human NMDA receptor rather than rodent NMDA receptor are used.
  • rat NMDA receptor subunits when overexpressed in a eukaryotic cell line, in particular a human cell line, have a higher propensity to accumulate outside the cell expressing the subunit in the form of protrusion-like or spherical aggregates made of unfolded protein which may be bound and stained by a secondary antibody with a detectable dye in a non-specific manner. Therefore, such aggregates may obscure stained cells or may be confused with stained cells the presence of which normally indicates that the sample is positive.
  • the mammalian cell is a cell line from a mammal, preferably from a human.
  • Exemplary cells lines include, but are not limited to HEK293, T-Rex-HeLa, T-Rex- CHO and T-RExTM-Jurkat cells and derivatives thereof.
  • the term “human NMDAR receptor”, as used herein, refers to a receptor comprising human NR1 subunits or the combination of an NR1 subunit with another receptor subunit such as NR2. It is essential that the NR1 subunit is present, since it comprises the autoantibody binding site.
  • NR1 subunits when expressed in a mammalian cell, preferably in the presence of a less than equivalent amount of another receptor subunit such as NR2, have the ability to form NR1 homodimers, which are also capable to binding to the autoantibody.
  • the human NR1 subunit has the sequence shown in SEQ ID N01.
  • the human NR2 subunit has the sequence shown in SEQ ID N02 or SEQ ID N04.
  • polypeptides may not only be carried out using the polypeptides, in particular a polypeptide comprising the native sequence of a polypeptide such as the NR1 subunit of the human NMDA receptor or nucleic acids having the exact sequences referred to in this application explicitly, for example by function, name, sequence or accession number, or implicitly, but also using variants of such polypeptides or nucleic acids.
  • the term “variant”, as used herein, may refer to at least one fragment of the full length sequence referred to, more specifically one or more amino acid or nucleic acid sequence which is, relative to the full-length sequence, truncated at one or both termini by one or more amino acids.
  • a fragment comprises or encodes for a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 850, 870, 890, 900, 910, 920 or 930 successive amino acids of the original sequence or a variant thereof.
  • the total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 750, 850, 870, 890, 900, 910, 920 or 930 or more amino acids.
  • variant relates not only to at least one fragment, but also to a polypeptide or a fragment thereof comprising amino acid sequences that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99, 99,3, 99,4, 99,5, 99,6, 99,7, 99,8 or 99,9, preferably at least 99,3 % identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity, for example the ability of an antigen to bind to an (auto)antibody, or the fold or structure of the polypeptide are deleted or substituted and/or one or more such essential amino acids are replaced in a conservative manner and/or amino acids are added such that the biological activity of the polypeptide is preserved.
  • the state of the art comprises various methods that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity, see for example Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3 rd edition.
  • the ClustalW software (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007).
  • Clustal W and Clustal X version 2.0 Clustal W and Clustal X version 2.0.
  • polypeptide and variants thereof may, in addition, comprise chemical modifications, for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
  • chemical modifications for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
  • Any modification is designed such that it does not abolish the biological activity of the variant.
  • variants may also be generated by N- or/and C-terminal fusion of polypeptides, fragments or variants thereof with other known polypeptides or variants thereof and comprise active portions or domains, preferably having a sequence identity of at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % when aligned with the active portion of the reference sequence, wherein the term "active portion”, as used herein, refers to an amino acid sequence, which is less than the full length amino acid sequence or, in the case of a nucleic acid sequence, codes for less than the full length amino acid sequence, respectively, and/or is a variant of the natural sequence, but retains at least some of the biological activity.
  • the term "variant" of a nucleic acid comprises nucleic acids the complementary strand of which hybridizes, preferably under stringent conditions, to the reference or wild type nucleic acid.
  • Stringency of hybridization reactions is readily determinable by one of ordinary skilled in the art, and in general is an empirical calculation dependent on probe length, washing temperature and salt concentration. In general longer probes require higher temperatures for proper annealing, while shorter probes less so.
  • Hybridization generally depends on the ability of denatured DNA to reanneal to complementary strands present in an environment below their melting temperature: The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which may be used.
  • stringent conditions are applied for any hybridization, i.e. hybridization occurs only if the probe is 70 % or more identical to the target sequence.
  • Probes having a lower degree of identity with respect to the target sequence may hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example lowering the concentration of salt to 2 x SSC or, optionally and subsequently, to 0,5 x SSC, while the temperature is, in order of increasing preference, approximately 50°C - 68°C, approximately 52°C - 68°C, approximately 54°C - 68°C, approximately 56°C - 68°C, approximately 58°C - 68°C, approximately 60°C - 68°C, approximately 62°C - 68°C, approximately 64°C - 68°C, approximately 66°C - 68°C.
  • the temperature is approximately 64°C - 68°C or approximately 66°C - 68°C. It is possible to adjust the concentration of salt to 0.2 x SSC or even 0.1 x SSC. Nucleic acid sequences having a degree of identity with respect to the reference or wild type sequence of at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % may be isolated.
  • the term variant of a nucleic acid sequence refers to any nucleic acid sequence that encodes the same amino acid sequence and variants thereof as the reference nucleic acid sequence, in line with the degeneracy of the genetic code.
  • the variant of the polypeptide has biological activity.
  • biological activity is the ability to bind specifically to an autoantibody binding to the NMDA receptor, preferably human NMDA receptor, as found in a patient suffering from an autoimmune disease associated with such autoantibody, preferably associated with a neurological disease such as epilepsy, encephalitis and PNS.
  • whether or not a variant of the polypeptide has such biological activity may be checked by determining whether or not the variant of interest binds to an autoantibody from a sample of a patient which autoantibody binds to wild type human NMDA receptor, preferably as determined by indirect immunofluorescence as described in the experimental section of this application.
  • At least one NR1 subunit remains that comprises amino acids 25-380 of the NR1 subunit, because the inventors of US7,972,976 have shown that this is the part of the antigen that is essential for recognition of the autoantibody. Further guidance based on site-directed mutagenesis may be found by Gleichman et al. (2012) who identified an epitope comprising N368 and G369 (Gleichman, A.J., Spruce, L. A., Dalmau, J., Seeholzer, S. H., and Lynch, D. R. (2012) Anti-NMDA Receptor Encephalitis Antibody binding Is Dependent on Amino Acid Identity of a Small Region within the GluN1 Amino Terminal Domain, J.
  • the person skilled in the art when designing variants or fragments, will consider that one or more amino acid residues characteristic of the human sequences should be preserved.
  • the variant comprises one or more, preferably at least two, three, four, five, or six, more preferably all from the group comprising Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415, with reference to SEQ ID N01 and highlighted in Fig. 6., while preferably other amino acid residues, as far as not essential for the biological activity or use for the detection of an autoantibody to NR1, may be modified or may be absent.
  • the cell according to the present invention overexpresses a polypeptide comprising human NMDA receptor or a variant.
  • the term “overexpressing”, as used herein means that the cell has been transfected with a nucleic acid, either transiently or stably in the sense that the nucleic acid has been incorporated in the genome of the cell, that comprises a nucleic acid sequence encoding a polypeptide comprising the human NMDA receptor or a variant thereof under the control of a promotor. Consequently, the transfected cell expresses more polypeptide recognized by the autoantibody than the same type of cell normally would, probably at least 10, 20, 30, 50, 100, 200 or 500 % more as judged by quantitative Western Blot.
  • the promotor may be an inducible promotor, which allows for the induction of expression by addition of an inducer.
  • the person skilled in the art is familiar with protocols and vectors for transiently overexpressing a polypeptide in a eukaryotic cell, for example the pTriEx system from Novagen and with protocols and vectors for stably transfecting a eukaryotic cell, for example the pcDNATM4/TO vector system from Invitrogen.
  • a fixed mammalian cell may be used.
  • the term “fixed” cell refers to a cell that has been treated with a reactive chemical compound to the effect that the cell is no longer metabolically active, but still presents its epitopes for immunostaining with antibodies and their subsequent detection, for example by fluorescence.
  • the reactive chemical compound is selected from the group comprising acetone, formalin, methanol and ethanol or mixtures thereof, preferably all of them.
  • the person skilled in the art is familiar with protocols that may be used to prepare fixed cells. Essentially, the cell which is attached to a solid support is washed by using washing buffer, followed by contacting with the reactive compound, for example immersion. Pure acetone or formalin or aqueous dilutions of the reactive chemical compound may be used.
  • the cell is on a carrier for microscopic immunofluorescence analysis.
  • a carrier may be a glass slide.
  • the cell on the glass slide may be covered with a mounting buffer.
  • a mounting medium is a liquid which helps maintain a near physiological pH to maintain the molecular structure of any diagnostically relevant molecular and their epitopes, is compatible with the emission of a fluorescence signal and prevents a premature loss of fluorescence due to bleaching of the fluorophore.
  • its optical properties are matched with other buffers used, in particular it’s the refractive index which allows for an efficient microscopic fluorescence analysis.
  • the mounting medium comprises a base component, preferably selected from the group comprising water, glycerol, natural oil or plastic or a mixture thereof, preferably water and glycerol. It may further comprise an antifade constituent which may reduce bleaching, preferably selected from the group comprising NPG (N-propyl gallate), DABCO (1,4- diazabicyclo[2.2.2]octane), 4POBN ((4-Pirydyl-1-oxide)-N-tert-butyl nitrone) and PPD (P- phenylanediamine).
  • NPG N-propyl gallate
  • DABCO 1,4- diazabicyclo[2.2.2]octane
  • 4POBN ((4-Pirydyl-1-oxide)-N-tert-butyl nitrone)
  • PPD P- phenylanediamine
  • a cover glass may be placed on top of the composition comprising the sample and the mounting medium.
  • Slides with cover glasses (FB 112d-1005-1) are available from EUROIMMUN Medizinische Labordiagnostika, AG.
  • any carrier compatible with microscopic analysis of the fluorescence pattern may be used.
  • the carrier may comprise a mock-transfected cell, which has been transfected with the same vector as the cell overexpressing a polypeptide comprising NMDA receptor, but without the nucleic acid encoding for the latter. Such mock-transfected cell may serve as a negative control.
  • the carrier is configured for analysis using an immunofluorescence microscope.
  • the carrier may comprise a field comprising the cell according to the invention.
  • the carrier may comprise additional fields.
  • the fields are preferably surrounded by a hydrophobic surface.
  • Each of these fields may comprise a cell overexpressing another autoantigen, preferably selected from the group comprising GABA A, GABA B, Lgl 1 , AMPAR1/R2, CASPR2, DPPX, Hu, Ma2, CV2, amphiphysin or a variant thereof.
  • Suitable autoantigens and their sequences and variants are described in US15/023131 (GABA A), US8685656 (GABA B), US9250250 (Lgl1), Lai, M., Hughes, E. G., Peng, X., Zhou, L, Gleichman, A.
  • AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location, Ann. Neurol. 65(4), 424-434 (AMPAR1/R2), US9188587 (CASPR2), US9719993 (DPPX), US6193948/US5603934/US5807705 (Hu), US7026450 (Ma2),
  • the cell according to the invention is preferably spatially separated from any of the mock-transfected cell or a cell expressing another autoantigen such that a fluorescence signal can be assigned to a specific autoantigen, for example the human NMDA receptor.
  • the carrier may comprise one or more additional fields, each comprising a tissue from the group comprising mammalian hippocampus and/or mammalian cerebellum, more preferably rat hippocampus rat cerebellum, primate hippocampus and primate cerebellum, most preferably all of them.
  • the cell is transiently transfected, which preferably means that the cell is transfected with a vector comprising a nucleic acid encoding for a polypeptide comprising human NMDA receptor, but this nucleic acid is not integrated into the genome of the cell. Consequently, the cell may lose the vector when cultivated for a prolonged time to the effect that it no longer expresses said polypeptide.
  • the cell permanently expresses the human NMDA receptor, more preferably because a nucleic acid encoding for a polypeptide comprising the NMDA receptor has been integrated into the genome. Consequently, the cell is likely to express the polypeptide permanently.
  • a secondary antibody is an antibody binding specifically to all antibodies from an antibody class, preferably preferably IgG. Secondary antibodies typically recognize the constant domain of said class, but may also recognize other epitopes shared by antibodies from the class of interest, for example a conformational epitope across the 3D structure. A wide range of them is commercially available, for example from Thermo Fisher. It may be a monoclonal or a polyclonal antibody.
  • a secondary antibody may bind specifically to all isotypes from the antibody class.
  • a secondary antibody to IgG class antibodies may bind to lgG1, lgG2, lgG3 and lgG4 isotypes. This may be achieved by using as a secondary antibody to the class, preferably to IgG class antibodies, a mixture comprising an antibody binding specifically to each IgG isotype or a single antibody which reacts with all isotypes of interest.
  • the use of secondary antibodies is explained in Kruger, N. J., Detection of Polypeptides on Blots Using Secondary Antibodies, in The Protein Protocols Handbook (ed. J. M. Walker), page 967, volume 1996, Springer.
  • the autoantibody to be detected or a secondary antibody used binds preferably specifically to human NMDA receptor or the autoantibody to be detected, respectively.
  • Binding specifically preferably means that the binding reaction is stronger than a binding reaction characterized by a dissociation constant of 1 x 10 5 M, more preferably 1 x 10 7 M, more preferably 1 x 10 8 M, more preferably 1 x 10 9 M, more preferably 1 x 10 10 M, more preferably 1 x 10 11 M, more preferably 1 x 10 12 M, as determined by surface plasmon resonance using Biacore equipment at 25 °C in PBS buffer at pH 7.
  • the cell is bound to an autoantibody to human NMDA receptor, and a secondary antibody is bound to the antibody.
  • the secondary antibody recognizes IgG class antibodies.
  • the secondary antibody may comprise a detectable fluorescent label, more preferably FITC (fluorescein isothiocyanate).
  • the method according to the present invention comprises the step providing the carrier according to the present invention.
  • the carrier may then be contacted with the sample suspected of comprising the autoantibody under conditions allowing for binding of any autoantibodies to the cell and the NMDA receptor or variant thereof expressed by the cell.
  • the sample may then be removed and the carrier with the cell may be washed to remove any remaining sample.
  • a secondary autoantibody or similar reagent or means binding to the autoantibody and carrying a detectable label such as a fluorescent dye may then be contacted with the carrier under conditions allowing formation of a complex between any bound autoantibody and the secondary antibody.
  • the carrier may be washed then to remove non-bound secondary antibody.
  • the presence of the autoantibody is detected by checking whether the secondary antibody may be detected, preferably by immunofluorescence, more preferably emitted by fluorescein or a derivative thereof, most preferably FITC.
  • the cell, method, kit, polypeptide or carrier is used for the diagnosis of a disease, preferably neurological autoimmune disease, more preferably from the group comprising paraneoplastic syndrome, autoimmune encephalitis, limbic encephalitis and epilepsy.
  • a disease preferably neurological autoimmune disease, more preferably from the group comprising paraneoplastic syndrome, autoimmune encephalitis, limbic encephalitis and epilepsy.
  • the cell, method, kit, polypeptide or carrier is used for the diagnosis of teratoma.
  • the term ..diagnosis is to be used in its broadest possible sense and may to any kind of procedure aiming to obtain information instrumental in the assessment whether a patient suffers or is likely or more likely than the average or a comparative subject, the latter preferably having similar symptoms, to suffer from certain a disease or disorder in the past, at the time of the diagnosis or in the future, to find out how the disease is progressing or is likely to progress in the future or to evaluate the responsiveness of a patient or patients in general with regard to a certain treatment, for example the administration of immunosuppressive drugs, or to find out whether a sample is from such a patient.
  • Such information may be used for a clinical diagnosis, but may also be obtained by an experimental and/or research laboratory for the purpose of general research, for example to determine the proportion of subjects suffering from the disease in a patient cohort or in a population.
  • diagnosis comprises not only diagnosing, but also prognosticating and/or monitoring the course of a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or candidate drug, for example to determine its efficacy. While the result may be assigned to a specific patient for clinical diagnostic applications and may be communicated to a medical doctor or institution treating said patient, this is not necessarily the casefor other applications, for example in diagnostics for research purposes, where it may be sufficient to assign the results to an sample from an anonymized patient.
  • the methods and products according to the present invention may be used for interaction studies, including determining whether a drug candidate or other compound may interfere with the binding of an autoantibody to the NMDA receptor or may affect any downstream process or the strength of their binding to their target such as the NR1 subunit.
  • they may be used for monitoring the immune response, more preferably the emergence and/or titer of antibodies to NMDAR, following the administration of an immunogenic composition comprising NMDAR or an immunogenic variant thereof, for example to a mammal, which may be a mammal other than a human such as a laboratory animal.
  • the mere detection of the autoantibody in other words determining whether or not detectable levels of the antibody are present in the sample, is sufficient for the diagnosis. If the autoantibody can be detected, this will be information instrumental for the clinician’s diagnosis and indicates an increased likelihood that the patient suffers from a disease.
  • a clinician does usually not conclude whether or not the patient suffers or is likely to suffer from a disease, condition or disorders solely on the basis of a single diagnostic parameter, but needs to take into account other aspects, for example the presence of other autoantibodies, markers, blood parameters, clinical assessment of the patient’s symptoms or the results of medical imaging or other non- invasive methods such as polysomnography, to arrive at a conclusive diagnosis.
  • Other autoantibodies, markers, blood parameters, clinical assessment of the patient’s symptoms or the results of medical imaging or other non- invasive methods such as polysomnography to arrive at a conclusive diagnosis.
  • any symptoms or diseases referred to throughout this application is in line with the person skilled in the art’s understanding as of the filing date or, preferably, earliest priority date of this application as evidenced by text books and scientific publications. It should be mentioned that the inventive methods or uses or products, taken alone, cannot be used to arrive at a definite, final diagnosis.
  • the term “diagnosis” may also refer to a method or agent used to distinguish between two or more conditions associated with similar or identical symptoms, for example autoimmune encephalitis and an encephalitis caused by an infection.
  • the presence or absence of an antibody to a viral antigen may be determined according to the present invention, for example relating to a virus causing encephalitis.
  • the term “diagnosis” may also refer to a method or agent used to choose the most promising treatment regime for a patient.
  • the method or agent may relate to selecting a treatment regimen for a subject.
  • the detection of autoantibodies may indicate that an immunosuppressive therapy is to be selected, which may include administrating to the patient one or more immunosuppressive drugs.
  • any information or data demonstrating the presence of absence of the autoantibody may be communicated to the patient or a medical doctor treating the patient, preferably by telephone, in a written form or via the internet, for example as an email or text message.
  • the term dormitory autoantibody refers to an antibody binding specifically to an endogenous molecule of the animal, preferably mammal, more preferably human, which produces said autoantibody, wherein the level of such antibody is more preferably elevated compared to the average healthy subject.
  • the autoantibody is an autoantibody binding to mammalian, preferably human NMDA receptor.
  • the autoantibody may have the sequence of an antibody’s constant regions from the animal, preferably human, making it, but the variable region is able to bind specifically to the endogenous molecule of the animal, more specifically NMDA receptor.
  • the autoantibody is isolated and/or purified from a sample, preferably tissue, serum, plasma, blood or CSF from the animal, preferably human.
  • the autoantibody is a polyclonal, native antibody from the animal rather than a synthetic or recombinant antibody.
  • the method according to the present invention is preferably an in vitro method.
  • a kit comprising the cell or the carrier and further comprising one or more, preferably all reagents from the group comprising a secondary antibody, preferably labeled with a detectable label, a washing solution, a positive control, a negative control, a detergent, a cover glass, a mounting medium and a physiological salt solution, preferably PBS, or salt required to prepare it.
  • the positive control is a diluted sample, preferably serum or CSF, from a patient suffering from NMDA receptor autoimmune encephalitis or a monoclonal antibody to human NMDA receptor, preferably binding to the NR1 subunit recognized by the secondary antibody.
  • the negative control may be a diluted sample from a healthy subject, for example a blood donor.
  • the kit may comprise instructions how to carry out the assay.
  • the secondary antibody is a secondary antibody to IgG class antibodies, preferably human IgG glass antibodies.
  • the present invention provides a use of the cell, the polypeptide, the carrier for the manufacture of kit a composition for the diagnosis of a disease.
  • any method or use according to the present invention may be intended for a non-diagnostic use, i.e. determining the presence of an autoantibody to binding to human NMDA receptor for a use other than diagnosing a patient.
  • the method or use may be for testing in vitro the efficiency of a medical device designed to remove an autoantibody from a patient’s blood, wherein the testing is performed on a liquid other than patient’s blood.
  • its capacity to remove autoantibody may be checked by running a solution comprising antibody to human NMDA receptor through the device, followed by use of the method according to the present invention to confirm that less or no antibody is in the solution that has been passed through the device, i.e. showing that the device has still the capacity to remove antibody from the solution.
  • the method may be for confirming the reliability of a diagnostic assay and may involve detecting an antibody to human NMDA receptor in a solution, which is not a sample from a patient who requires a diagnosis, but is known to comprise an antibody to human NMDA receptor, preferably at a known concentration.
  • a solution which is not a sample from a patient who requires a diagnosis, but is known to comprise an antibody to human NMDA receptor, preferably at a known concentration.
  • it may be a recombinant antibody or a sample diluted in a dilution buffer such as PBS from an anonymous patient whose identity cannot be traced back.
  • the solution may be a negative control not comprising the antibody to check the background.
  • any method or use according to the present invention may be intended for generating an autoantibody profile, preferably for detecting a disease in a mammal, preferably a human.
  • any method or use according to the present invention may be for identifying a subject at risk of suffering from or developing a disease and/or a tumor.
  • the method may be for detecting an antibody, preferably autoantibody in a solution which is not a sample from a mammal to be diagnosed or for the purpose of providing a diagnosis, in particular not a diagnosis of a neurological disease such as NMDA receptor encephalitis.
  • the present invention provides an apparatus for analyzing a sample from a patient to detect an autoantibody against human NMDA receptor, indicating an increased likelihood of a disease or of developing a disease, comprising: a. a carrier, which contains a means for capturing the autoantibody from the sample when the sample is contacted with the carrier, wherein the means is the cell and the carrier is the carrier according to the present invention, b. a detectable means capable of binding to the antibody captured by the carrier when the detectable means is contacted with the carrier, wherein the detectable means is preferably a labeled secondary antibody capable of binding to the autoantibody captured on the carrier, c.
  • a means for removing any sample from the carrier and the detectable means optionally a means for removing any sample from the carrier and the detectable means, preferably by washing; d. a detecting device for detecting the presence of the detectable means and converting the results into an electrical signal, for example a fluorescence reader connected with a software capable of recognizing a pattern characteristic of a stained cell overexpressing human NMDAR in an image of the cell taken by the fluorescence reader or camera, and optionally a means for receiving the electronical signal from the detecting device and determining if the level of the signal is indicative of an increased likelihood of having or developing a disease, by comparing with the patterns characteristic of wild type or non- stained cells, preferably by a mock-transfected cell or cells not positively stained by an autoantibody binding to human NMDA receptor on the same carrier, or an input reference value obtained with samples from healthy subjects or by comparing the level of signal obtained with one sample with the level of signal obtained with a second sample obtained at a later time point, preferably at least one month
  • Fig. 1A shows immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding rat NR1 subunit of the NMDA receptor, stained with a sample from a patient suffering from NMDA receptor encephalitis.
  • White arrows mark artefacts in the form of protrusions which are artefacts resulting from the immunostaining of NR1 aggregates.
  • Fig. 1B shows immunofluorescence analysis of HEK293 cells permanently transfected with the human NR1 subunit. These images are clearly free of artefacts.
  • Fig. 2A shows immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding NR1 subunit of the rat NMDA receptor, stained with a 1:100 diluted sample from a patient who does not suffer from NMDA receptor encephalitis. Spherical bodies comprising NR1 aggregates are stained and could be mistaken for positively stained cells.
  • Fig. 2B shows immunofluorescence analysis of HEK293 permanently transfected with a vector encoding NR1 subunit of the human NMDA receptor, stained with the same sample. No spherical artefacts can be observed.
  • Fig. 3A shows immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding NR1 subunit of the rat NMDA receptor, stained with a sample from a patient who suffers from NMDA receptor encephalitis.
  • Spherical bodies comprising NR1 aggregates are stained and obscure stained positive cells, which are shown in Fig. 3B where the same sample was used to stain HEK293 cells permanently expressing NR1 of the human NMDA receptor.
  • Figs. 4A, 4B and 4C show immunofluorescence analysis of HEK293 cells stained with a 1:10 sample from a patient suffering from NMDA receptor encephalitis.
  • the cells transiently express rat NR1 (Fig. 4A), transiently human NR1 (Fig. 4B) or permanently human NR1 (Fig. 4C).
  • Fig. 4A protrusion-like and spherical artefacts can be observed.
  • Fig. 4B there are spherical artefacts, but the protrusions are less pronounced.
  • Fig. 4C there are no artefacts.
  • Figs. 5A, 5B and 5C show immunofluorescence analysis of HEK293 cells stained with a 1:10 sample from a patient not suffering from NMDA receptor encephalitis.
  • the cells transiently express rat NR1 (Fig. 5A), transiently human NR1 (Fig. 5B) or permanently human NR1 (Fig. 5C).
  • Fig. 5A spherical artefacts convey the impression of cells positively stained by autoantibodies to NMDAR.
  • Fig. 5B the spherical artefacts are less pronounced.
  • Fig. 5C there are none.
  • Fig. 6 shows an alignment of the amino acid sequences of human NR1 subunit depressionQuery 1“), which is SEQ ID N01, and rat NR1 subunit wellbeingSbjct 1“). Amino acids that differ are in bold in the human sequence.
  • the present invention comprises a range of novel nucleic acid and polypeptide sequences, more specifically
  • SEQ ID N01 polypeptide sequence human NR1: mstmrlltlallfscsvaraacdpkivnigavlstrkheqmfreavnqankrhgswkiqlnatsvthkpnaiqma lsvcedlissqvyailvshpptpndhftptpvsytagfyripvlglttrmsiysdksihlsflrtvppyshqssv wfemmrvyswnhiillvsddhegraaqkrletlleereskaekvlqfdpgtknvtallmeakelearviilsase ddaatvyraaamlnmtgsgyvwlvgereisgnalryapdgilglqlingknesahisdavgvvaqavhelleken itdpprgc
  • SEQ ID N02 (polypeptide sequence NMDAR subunit NR2a) mgrvgywtllvlpallvwrgpapsaaaekgppalniavmlghshdvterelrtlwgpeqaaglpldvnvvallmn rtdpkslithvcdlmsgarihglvfgddtdqeavaqmldfisshtfvpilgihggasmimadkdptstffqfgas iqqqatv lkimqdydwhvfslvttifpgyrefisfvkttvdnsfvgwdmqnvitldtsfedaktqvqlkkihss villycskdeavlilsearslgltgydffwivpslvsgntelipkefpsglisvsyddwd
  • the methodology and reagents used are according to the manufacturer’s instruction in the 11 FT Neurology Mosaics (EUROIMMUN Medizinische Labordiagnostika AG, product number FA112d-1.
  • the test system centers around the incubation of combinations of substrates with diluted patient sample. If the reaction is positive, specific antibodies of classes IgA, IgG and IgM attach to the antigens.
  • the attached antibodies are stained with FITC- labelled anti-human antibodies and made visible with a fluorescence microscope. Briefly, human serum samples were diluted 1:10 or 1:100 in PBS-Tween, followed by vortexing for 2 seconds.
  • the carrier with the fields is covered with up to 10 mI of mounting medium per field and a glass cover slide, followed by fluorescence microscopy analysis using a EUROSTAR microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck.
  • HEK293 cells transiently expressing NR1 subunit from the human receptor produces spherical bodies, but considerably less pronounced protrusions.
  • HEK293 cells permanently overexpression NR1 from the human receptor produces neither artefact. Confusion of the staff analyzing the images and false-positive results can be avoided by using these cells.

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Abstract

La présente invention concerne un support pour l'analyse par immunofluorescence microscopique recouvert d'une cellule eucaryote surexprimant un polypeptide comprenant le récepteur NMDA humain ou une variante de celui-ci, un support comprenant la cellule et une cellule transfectée par simulacre, la cellule et la cellule transfectée par simulacre étant séparées dans l'espace, un kit comprenant la cellule ou le support et une utilisation de la cellule ou du support ou d'un polypeptide comprenant le récepteur NMDA humain ou une variante de celui-ci pour produire un signal dépourvu d'artefact pour la détection d'un auto-anticorps du récepteur NMDA humain.
EP20767572.9A 2019-09-13 2020-09-10 Détection améliorée des auto-anticorps du récepteur nmda Pending EP4028769A1 (fr)

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