WO2024258729A2

WO2024258729A2 - Assessing and treating paraneoplastic neurologic syndrome

Info

Publication number: WO2024258729A2
Application number: PCT/US2024/032765
Authority: WO
Inventors: Divyanshu DUBEY; Sean J. Pittock; John R. Mills; Surendra Dasari; Andrew M. KNIGHT; Mohamed Rezk
Original assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Current assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Priority date: 2023-06-12
Filing date: 2024-06-06
Publication date: 2024-12-19
Anticipated expiration: 2025-12-12
Also published as: EP4724811A2; WO2024258729A9; WO2024258729A3

Abstract

This document provides methods and materials for assessing and/or treating mammals having a paraneoplastic neurologic syndrome (PNS). For example, methods and materials for using a Sloan Kettering virus family transcriptional corepressor 2 (SKOR2) polypeptide and/or one or more fragments of a SKOR2 polypeptide to detect the presence or absence of autoantibodies present in immune-mediated PNS are provided.

Description

ASSESSING AND TREATING PARANEOPLASTIC NEUROLOGIC SYNDROME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 63/472,427, filed June 12, 2023. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated in its entirety into this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2224 W0_SL.xml.” The XML file, created on May 22, 2024, is 3,833 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to methods and materials involved in assessing and/or treating mammals having a paraneoplastic neurologic syndrome (PNS). For example, this document provides methods and materials for using a Sloan Kettering virus family transcriptional corepressor 2 (SKOR2) polypeptide and/or one or more fragments of a SKOR2 polypeptide to detect the presence or absence of autoantibodies present in immune-mediated PNS.

BACKGROUND

PNSs are group of disorders affecting one or multiple levels of the neuroaxis associated with underlying tumors (Graus et al., Neurol. Neuroimmunol. Neuroinflamm., 8(4):el014 (2021)). An antibody or autoantigen-specific cell-mediated immune response against neural antigen expressed in the tumor is the potential etiology for these rare but refractory disorders (Ganaraja et al., Neurol. Sci., 43:3583-3594 (2022)).

SUMMARY

This document provides methods and materials for assessing and/or treating mammals (e.g., humans) having a PNS. For example, this document provides methods and materials for detecting autoantibodies in mammals (e.g., humans) having a PNS. As described herein, a specific IgG autoantibody can be found in serum of some individuals having a PNS (also referred to herein as PNS-specific autoantibodies). Also as described herein, PNS-specific autoantibodies can target and bind to a SK0R2 polypeptide. Accordingly, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide can be used to determine if a sample obtained from a mammal (e.g., a human) having a PNS contains PNS-specific autoantibodies (e.g., anti-SKOR2 polypeptide antibodies), and the presence of PNS-specific autoantibodies (e.g., anti-SKOR2 polypeptide antibodies) can be used to identify the mammal as having a PNS. Serological detection of PNS-specific autoantibodies (e.g., anti-SKOR2 polypeptide antibodies) can allow for earlier diagnosis of a PNS and can thus allow clinicians to provide appropriate treatment of a PNS.

In general, one aspect of this document features methods for determining whether or not a mammal has an immune-mediated PNS. The methods can include, or consist essentially of, (a) contacting a sample from a mammal with a composition including an antigen to form an antigen-anti- SK0R2 polypeptide autoantibody complex if the sample includes an anti- SK0R2 polypeptide autoantibody, where the antigen is a SK0R2 polypeptide, a fragment of the SK0R2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to the anti- SK0R2 polypeptide autoantibody; and (b) detecting the presence or absence of the complex, where the presence of the complex indicates that the mammal has the immune-mediated PNS, and where the absence of the complex indicates that the mammal does not have the immune-mediated PNS. The composition can include a cell lysate obtained from a cell (a) including exogenous nucleic acid encoding the antigen and (b) expressing the antigen. The antigen can be the SK0R2 polypeptide. The antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2. The antigen can be covalently linked to a detectable label. The detectable label can be a green fluorescent protein (GFP) polypeptide, tetramethylrhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), a poly(His) tag, a glutathione-S-transferase (GST) tag, biotin, a Flag tag, or a myc tag. The detecting can include performing an immunological assay. The mammal can be a human. The sample can be a serum sample. The method can include detecting the presence of the complex. The method can include classifying the mammal as having the immune-mediated PNS. The method can include detecting the absence of the complex. The method can include classifying the mammal as not having the immune-mediated PNS. The PNS can be cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonus-myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, or dysautonomia.

In another aspect, this document features methods for treating a mammal having an immune-mediated PNS. The methods can include, or consist essentially of, (a) determining that a sample from a mammal can include the presence of anti-SKOR2 polypeptide autoantibodies, and (b) administering an immunosuppressant to the mammal. The determining step can include: (i) contacting the sample with a composition including an antigen to form an antigen-anti- SKOR2 polypeptide autoantibody complex if the sample includes an anti-SKOR2 polypeptide autoantibody, where the antigen can be a SKOR2 polypeptide, a fragment of the SKOR2 polypeptide having the ability to bind to the anti- SK0R2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody; and (ii) detecting the presence of the complex, thereby determining that the mammal has the presence of the anti-SKOR2 polypeptide autoantibodies. The composition can include a cell lysate obtained from a cell (a) including exogenous nucleic acid encoding the antigen and (b) expressing the antigen. The antigen can be the SK0R2 polypeptide. The antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2. The antigen can be covalently linked to a detectable label. The detectable label can be a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, or a myc tag. The mammal can be a human. The sample can be a serum sample. The PNS can be cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonus-myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, or dysautonomia. The immunosuppressant can be rituximab, mycophenolate, cyclophosphamide, or azathioprine.

In another aspect, this document features methods for treating an immune-mediated PNS. The methods can include, or consist essentially of, administering an immunosuppressant to a mammal having a PNS and that was identified as having anti- SK0R2 polypeptide autoantibodies. The identifying can include: (a) contacting a sample from the mammal with a composition including an antigen to form an antigen-anti- SKOR2 polypeptide autoantibody complex if the sample includes an anti-SKOR2 polypeptide autoantibody, where the antigen can be a SK0R2 polypeptide, a fragment of the SK0R2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to the anti-SK0R2 polypeptide autoantibody; and (b) detecting the presence of the complex, thereby identifying the mammal as having the anti-SKOR2 polypeptide autoantibodies. The composition can include a cell lysate obtained from a cell (a) including exogenous nucleic acid encoding the antigen and (b) expressing the antigen. The antigen can be the SK0R2 polypeptide. The antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2. The antigen can be covalently linked to a detectable label. The detectable label can be a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, or a myc tag. The mammal can be a human. The sample can be a serum sample. The PNS can be cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonus-myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, or dysautonomia. The immunosuppressant can be rituximab, mycophenolate, cyclophosphamide, or azathioprine.

In another aspect, this document features methods for treating a mammal having a PNS. The methods can include, or consist essentially of, (a) determining that a sample from a mammal lacks the presence of anti-SKOR2 polypeptide autoantibodies, and (b) administering a therapy for the PNS to the mammal, where the therapy is not an immunosuppressant. The method of claim 43, where the determining step can include: (i) contacting the sample with a composition including an antigen to form an antigen-anti- SK0R2 polypeptide autoantibody complex if the sample includes an anti-SKOR2 polypeptide autoantibody, where the antigen can be a SKOR2 polypeptide, a fragment of the SK0R2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody; and (ii) detecting the absence of the complex, thereby that the mammal lacks the presence of anti-SKOR2 polypeptide autoantibodies. The composition can include a cell lysate obtained from a cell (a) including exogenous nucleic acid encoding the antigen and (b) expressing the antigen. The antigen can be the SKOR2 polypeptide. The antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2. The antigen can be covalently linked to a detectable label. The detectable label can be a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, or a myc tag. The mammal can be a human. The sample can be a serum sample. The PNS can be cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonus-myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, or dysautonomia. The therapy for the PNS can be a physical therapy, speech therapy, cognitive behavioral therapy, or cognitive rehabilitation.

In another aspect, this document features methods for treating a PNS. The methods can include, or consist essentially of, administering a therapy for a PNS to a mammal that was identified as lacking anti-SKOR2 polypeptide autoantibodies, where the therapy is not an immunosuppressant. The identifying can include: (a) contacting a sample from the mammal with a composition including an antigen to form an antigen-anti- SKOR2 polypeptide autoantibody complex if the sample includes an anti-SKOR2 polypeptide autoantibody, where the antigen can be a SKOR2 polypeptide, a fragment of the SKOR2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to the anti-SKOR2 polypeptide autoantibody; and (b) detecting the absence of the complex, thereby identifying the mammal as lacking anti-SKOR2 polypeptide autoantibodies. The composition can include a cell lysate obtained from a cell (a) including exogenous nucleic acid encoding the antigen and (b) expressing the antigen. The antigen can be the SK0R2 polypeptide. The antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2. The antigen can be covalently linked to a detectable label. The detectable label can be a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, or a myc tag. The mammal can be a human. The sample can be a serum sample. The PNS can be cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonus-myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, or dysautonomia. The therapy for the PNS can be a physical therapy, speech therapy, cognitive behavioral therapy, or cognitive rehabilitation.

In another aspect, this document features methods for treating an immune-mediated PNS. The methods can include, or consist essentially of, (a) removing blood from a mammal having an immune-mediated PNS, thereby obtaining removed blood, (b) removing at least some anti-SKOR2 polypeptide autoantibodies from the removed blood, thereby obtaining processed blood, and (c) reintroducing the processed blood into the mammal. The step (b) can include contacting the removed blood with immobilized antigen, where the antigen can be a SKOR2 polypeptide, a fragment of the SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, or a variant of the SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody. The antigen can be the SK0R2 polypeptide. The antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2. The mammal can be a human.

In another aspect, this document features methods for treating a PNS. The methods can include, or consist essentially of, (a) removing blood from a mammal having the PNS that was identified as having anti-SKOR2 polypeptide autoantibodies, thereby obtaining removed blood, (b) removing at least some anti-SKOR2 polypeptide autoantibodies from the removed blood, thereby obtaining processed blood, and (c) reintroducing the processed blood into the mammal. The step (b) can include contacting the removed blood with immobilized antigen, where the antigen can be a SK0R2 polypeptide, a fragment of the SK0R2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody. The antigen can be the SKOR2 polypeptide. The the antigen can be the variant. The antigen can be the fragment. The fragment can consist of the amino acid sequence set forth in SEQ ID NO:2.

In another aspect, this document features uses of a composition including an antigen to treat an immune-mediated PNS, where the antigen can be a SK0R2 polypeptide, a fragment of the SK0R2 polypeptide having the ability to bind to an anti-SK0R2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to an anti- SK0R2 polypeptide autoantibody.

In another aspect, this document features uses of a composition including an antigen to treat an immune-mediated PNS, where the antigen can be a SK0R2 polypeptide, a fragment of the SK0R2 polypeptide having the ability to bind to an anti-SK0R2 polypeptide autoantibody, or a variant of the SK0R2 polypeptide having the ability to bind to an anti- SK0R2 polypeptide autoantibody, where the use can include removing at least some anti- SK0R2 polypeptide autoantibodies from the blood of a mammal having the immune- mediated PNS.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DR WINGS

Figures 1A-1D: SK0R2 IgG identification and confirmation. Figure 1A) Patient IgG demonstrated nuclear staining at the junction of Purkinje cell layer and granule layer of the cerebellum in indirect tissue immunofluorescence on cryosectioned mouse cerebellum (Al = 20x magnification, and A2 = 40x magnification). Figure IB). Dual immuno staining of cryosectioned mouse brain tissues by commercial SKOR2-specific rabbit IgG in Bl and by a patient IgG in B2 demonstrated colocalization. Nuclei were stained by 4',6-diamidino-2- phenylindole, and the merged image of Bl and B2 is in B3. Figure 1C) T etram ethylrhodamine-conjugated anti-human IgG of patient in C2 binding to recombinant SK0R2 protein-tagged expressed in the transiently transfected COS7 cells in Cl. The combined picture with stained nuclei is in C3. Figure ID) Western blot of COS7 cell lysate containing recombinant SK0R2 demonstrated binding of IgG in sera of both paraneoplastic neurologic syndrome cases (designated PNS SK0R2 IgG+) and SK0R2 commercial antibody (designated +) to an approximately 130-kD protein; no healthy control individual serum IgG (designated HC SK0R2 IgG-) bound.

Figures 2A-2C: SK0R2 IgG positive patient course of illness and clinical studies. Figure 2A) Graphical time course of events for case 1. Major events are indicated in black text. Figure 2B) FLAIR sequence of MRI showed hyperintensities in the left external capsule, insula and thalamus (Bl, arrowhead). Hyper-metabolic left upper lobe lesion (arrow) was observed, which was confirmed on biopsy, to be lung adenocarcinoma (B2). Post immunotherapy MRI showed resolution of findings observed in first MRI (B3, arrowhead). Recurrence of the left insular hyperintensity was seen (B4). MRI at last follow up demonstrated global volume loss (B5). Figure 2C) Graphical time course of events for case 2. Major events are indicated in black text.

Figures 3 A-3F. Expression of SK0R2 in the pulmonary adenocarcinoma. Figures 3 A and 3B) The lung biopsy from a pulmonary adenocarcinoma patient showed neoplastic cells (indicated with arrows) with high nuclear/cytoplasm (N/C) ratio (H/E stain, high power image in B). Figures 3C and 3D) Immunohistochemistry revealed variable cytoplasmic SKOR2 immunoreactivities in the neoplastic cells. Figures 3E and 3F) Negative control by omitting the primary antibody showed no signals on the consecutive section. Scale bars: 200 pm for A, C, and E; and 50 pm for B, D, and F.

Figure 4. An amino acid sequence of an exemplary human SKOR2 polypeptide (SEQ ID NO:1).

DETAILED DESCRIPTION

This document is based, at least in part, on the discovery that a specific IgG autoantibody can be found in serum of some individuals having an immune-mediated PNS, and the discovery that a SK0R2 polypeptide is the antigen target of the PNS-specific autoantibodies.

This document provides methods and materials for assessing and/or treating mammals (e.g., humans) having a PNS. For example, this document provides methods and materials for detecting PNS-specific autoantibodies (e.g., anti-SKOR2 polypeptide antibodies) in mammals (e.g., humans) having a PNS. In some cases, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide can be used to determine if a sample contains PNS-specific autoantibodies (e.g., anti-SKOR2 polypeptide antibodies). For example, a sample (e.g., a serum sample) obtained from a mammal (e.g., a human) having a PNS can be contacted with one or more fragments of a SK0R2 polypeptide such that a PNS- specific autoantibody (e.g., an anti-SKOR2 polypeptide antibody), if present, forms a complex with the fragment of the SK0R2 polypeptide (an antibody- SK0R2 fragment complex). In some cases, a sample (e.g., a serum sample) obtained from a mammal (e.g., a human) having a PNS can be contacted with cells designed to express a SKOR2 polypeptide and/or a lysate from cells designed to express a SK0R2 polypeptide such that a PNS-specific autoantibody (e.g., an anti-SKOR2 polypeptide antibody), if present, forms a complex with the SK0R2 polypeptide (an antibody-SKOR2 polypeptide complex). The presence of PNS- specific autoantibodies (e g., anti-SKOR2 polypeptide antibodies) can be used to identify a mammal (e.g., a human) as having an immune-mediated PNS. Also provided herein are materials and methods for treating mammals (e.g., humans) having a PNS based, at least in part, on the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS- specific autoantibodies) in a sample (e.g., a serum sample) obtained from the mammal.

Any appropriate mammal having a PNS can be assessed and/or treated as described herein. Examples of mammals that can have a PNS and can be assessed and/or treated as described herein include, without limitation, primates (e.g., humans and monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, rats, goats, and guinea pigs. For example, humans having a PNS can be assessed for the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) and, optionally, can be treated as described herein. In some cases, the methods and materials provided herein can be used to assess and/or treat an animal that is not a mammal. For example, the methods and materials provided herein can be used to assess and/or treat a bird (e.g., a chicken).

In some cases, a mammal (e.g., a human) that can be assessed and/or treated as described herein can have cancer. Examples of cancers that a mammal (e g., a human) that can be assessed and/or treated as described herein can have include, without limitation, lung cancers, gallbladder cancers, breast cancers, testicular cancers, thymic cancers, ovarian cancers, uterine/fallopian tube cancers.

A mammal (e.g., human) that can be assessed and/or treated as described herein can have any type of PNS. Examples of types of PNS that can be assessed and/or treated as described herein include, without limitation, cerebellar degeneration (e.g., cerebellar ataxia), limbic encephalitis, encephalomyelitis, opsoclonus-myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, and dysautonomia.

A mammal (e.g., human) that can be assessed and/or treated as described herein can have a PNS that affects any one or more locations within the mammal’s body. Examples of locations within a mammal’s body that can be affected by PNS include, without limitation, skeletal muscles, central nervous system, peripheral nerves, and the autonomic system.

Any appropriate sample from a mammal (e.g., human) having a PNS can be assessed for the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) as described herein. Examples of samples that can be assessed as described herein include, without limitation, blood samples (e.g., whole blood samples, serum samples, and plasma samples), CSF samples, and urine samples. In cases where a sample is a plasma sample, the plasma sample can be obtained by plasmapheresis. In some cases, one or more biological molecules can be isolated from a sample. For example, polypeptides (e.g., antibodies) can be isolated from a sample and can be assessed as described herein.

Any appropriate SKOR2 polypeptide can be used to detect the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in sample (e.g., a sample obtained from a mammal such as a human having a PNS). Examples of SK0R2 polypeptides that can be used to detect anti-SKOR2 polypeptide autoantibodies (e.g., PNS- specific autoantibodies) include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no. NM_001278063 (version NM_001278063.4) and accession no. NP_001264992 (version NP_001264992.1), and those set forth in The UniProt Knowledgebase (UniProtKB; see, e.g., The UniProt Consortium, Nucleic Acids Research, 51(D1):D523-D531 (2023)) at accession no. Q2VWA4. In some cases, a SKOR2 polypeptide can have an amino acid sequence set forth in SEQ ID NO: 1 (see, e.g., Figure 4).

Any appropriate fragment of a SKOR2 polypeptide can be used to detect an anti- SKOR2 polypeptide autoantibody (e.g., a PNS-specific autoantibody). A fragment of a SKOR2 polypeptide can be any appropriate length (e.g., can include any number of amino acids) provided that it retains the ability to bind to an anti-SKOR2 polypeptide autoantibody (e.g., a PNS-specific autoantibody). For example, a fragment of a SKOR2 polypeptide can be from about 5 amino acids in length to about 1015 amino acids in length (e.g., from about 5 to about 800, from about 5 to about 600, from about 5 to about 400, from about 5 to about 200, from about 5 to about 100, from about 100 to about 1015, from about 300 to about 1015, from about 500 to about 1015, from about 700 to about 1015, from about 50 to about 1000, from about 100 to about 900, from about 200 to about 800, from about 300 to about 700, from about 400 to about 600, from about 100 to about 300, from about 200 to about 400, from about 300 to about 500, from about 500 to about 700, from about 600 to about 800, or from about 700 to about 900 amino acids in length). In some cases, a fragment of a SKOR2 polypeptide fragment can have an amino acid sequence set forth in SEQ ID NO:2 (e.g., can consist of an amino acid sequence set forth in SEQ ID NO:2, can consist essentially of an amino acid sequence set forth in SEQ ID NO:2, or can comprise an amino acid sequence set forth in SEQ ID NO:2).

In some cases, a SKOR2 polypeptide or a fragment of a SKOR2 polypeptide can be a variant of an amino acid sequence set forth in any one of SEQ ID NOs: 1-2. For example, a variant of a SKOR2 polypeptide or a fragment of a SKOR2 polypeptide can consist of, consist essentially of, or comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, except that the variant polypeptide includes one, two, three, four, or five amino acid substitutions within the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1 or SEQ ID NO:2), has one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1 or SEQ ID NO:2), and/or has one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1 or SEQ ID NO:2), provided that the SKOR2 polypeptide or the fragment of a SKOR2 polypeptide retains the ability to bind to an anti- SKOR2 polypeptide autoantibody (e.g., a PNS-specific autoantibody).

Examples of SK0R2 polypeptides that can be used to detect anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) include, without limitation, polypeptides comprising SEQ ID NO: 1 and polypeptide comprising SEQ ID NO:2 as shown in Table 1. Table 1. Examples of SKOR2 polypeptides and variants thereof.

Any appropriate method can be used to obtain a SKOR2 polypeptide or a fragment of a SK0R2 polypeptide described herein (e.g., a polypeptide that consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, a polypeptide that consists essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 1-2, or a polypeptide that comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-2) or a variant thereof. In some cases, a SK0R2 polypeptide or a fragment of a SKOR2 polypeptide (or a variant thereof) can be obtained using polypeptide recombinant methods and/or synthesizing methods. For example, a polynucleotide sequence encoding a SK0R2 polypeptide (or a variant thereof) and/or a polynucleotide sequence encoding a fragment of a SKOR2 polypeptide (or a variant thereof) can be inserted into a plasmid or other vector that can then be delivered to host cells that can be induced to transcribe and translate the polynucleotide into the polypeptide. In some cases, a polynucleotide sequence for a full length polypeptide (e g., a full length SK0R2 polypeptide) can be inserted into host cells that can produce the full length polypeptide and then that full length polypeptide can be processed into a smaller polypeptide or a functional variant of interest (e.g., a fragment of a SK0R2 polypeptide).

A SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) can be provided in any appropriate context. In some cases, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) that can be used to detect the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) can be present in a cell lysate. For example, a SK0R2 polypeptide can be present in a cell lysate obtained from a cell having (e.g., designed to have) an exogenous nucleic acid encoding the SK0R2 polypeptide. For example, a fragment of a SK0R2 polypeptide can be present in a cell lysate obtained from a cell having (e.g., designed to have) an exogenous nucleic acid encoding the fragment of a SK0R2 polypeptide.

In some cases, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) that can be used to detect the presence or absence of anti- SK0R2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) can be present on a cell (e.g., an intact cell). For example, a SK0R2 polypeptide can be present on a cell having (e.g., designed to have) an exogenous nucleic acid encoding the SK0R2 polypeptide and expressing the SK0R2 polypeptide on its surface. For example, a fragment of a SK0R2 polypeptide can be present on a cell having (e.g., designed to have) an exogenous nucleic acid encoding the fragment of a SK0R2 polypeptide and expressing the fragment on its surface.

In some cases, a SKOR2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or a variant thereof) that can be used to detect the presence or absence of anti- SK0R2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) can be substantially pure. The term “substantially pure” as used herein with reference to a SKOR2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) refers to material which is substantially or essentially free from components (e.g., other polypeptides, lipids, carbohydrates, and nucleic acid) that normally accompany the material as it is found in its native state. Thus, substantially pure polypeptides as described herein do not contain at least some of the materials normally associated with the polypeptides in their in situ environment. For example, a substantially pure SK0R2 polypeptide and/or one or more substantially pure fragments of a SK0R2 polypeptide (or a variant thereof) can constitute the major component in a mixture of components (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more by weight).

In some cases, a SKOR2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or variant thereof) that can be used to detect the presence or absence of anti- SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) can be present in a composition. A composition including a SKOR2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or a variant thereof) that can be used to detect the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) can include any appropriate amount of the SKOR2 polypeptide and/or the one or more fragments of a SKOR2 polypeptide (or a variant thereof). For example, at least 5 percent (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more) of the polypeptide content of a composition including a SKOR2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or a variant thereof) can be the SKOR2 polypeptide and/or the one or more fragments of a SKOR2 polypeptide (or the variant thereof). In some cases, a composition including a SKOR2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or a variant thereof) can be enriched for the SK0R2 polypeptide and/or the one or more fragments of a SKOR2 polypeptide (or the variant thereof).

In some cases, a SKOR2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or variant thereof) described herein can lack any modification. For example, a SK0R2 polypeptide and/or one or more fragments of a SKOR2 polypeptide (or variant thereof) can be used to detect the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) without any modification. For example, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or variant thereof) that can be used to detect the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) can lack any detectable label.

In some cases, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) described herein can be modified. For example, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) that can be used for detecting the presence or absence of anti-SKOR2 polypeptide autoantibodies (e g., PNS-specific autoantibodies) can include (e.g., can be covalently linked to) one or more labels (e.g., one or more detectable labels). In some cases, a label can be a polypeptide tag (e.g., an affinity tag). In some cases, a label can be a fluorescent label. In some cases, a label can be covalently linked to a chemiluminescent label. In some cases, a label can have enzymatic activity. In some cases, a label can be radioactive. Examples of labels that can be attached to SK0R2 polypeptide or a fragment of a SK0R2 polypeptide (or a variant thereof) include, without limitation, green fluorescent protein (GFP) polypeptides, TRITC, fluorescein isothiocyanate (FITC), poly(His) tags, glutathione- S-transferase (GST) tags, biotin, Flag tags, and myc tags.

Any appropriate method can be used to detect the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample (e.g., a serum sample) obtained from a mammal (e.g., a human) having a PNS. For example, immunological assays using a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) provided herein can be used to determine if a sample contains anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies). In some cases, a sample (e g., a serum sample) obtained from a mammal (e.g., a human) having a PNS can be contacted with a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) described herein such that an anti-SKOR2 polypeptide autoantibody (e.g., a PNS-specific autoantibody), if present, forms a complex with the SK0R2 polypeptide and/or the one or more fragments of a SK0R2 polypeptide (or the variant thereof), and the presence or absence of that complex can be used to determine whether or not anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) are present in the sample. In some cases, an immobilized SK0R2 polypeptide and/or one or more immobilized fragments of a SK0R2 polypeptide (or a variant thereof) can be used to capture an anti-SKOR2 polypeptide autoantibody (e.g., a PNS-specific autoantibody) if present within a sample being tested, and an anti-Ig antibody (e.g., an anti-human IgG antibody when testing for human autoantibodies) can be used to determine whether or not anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) were captured. In some cases, an anti-Ig antibody can be labeled (e.g., fluorescently or enzymatically labeled) to aid in detection.

In some cases, the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample (e.g., a serum sample) obtained from a mammal (e.g., a human) having a PNS can be detected as described in Example 1.

In some cases, methods for assessing a sample (e.g., a serum sample) obtained from a mammal (e.g., a human) having a PNS for the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) as provided herein can be used to identify a mammal as having a PNS. For example, when a mammal (e.g., a human) having a PNS is identified as having a presence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample (e.g., a serum sample) obtained from the mammal, the mammal can be classified as having an immune-mediated PNS. In another example, when a mammal (e.g., a human) having a PNS is identified as lacking anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample (e.g., a serum sample) obtained from the mammal, the mammal can be classified as having a PNS that is not immune-mediated (or is not associated with SKOR2-specific autoantibodies).

This document also provides methods and materials for treating a mammal (e.g., a human) having a PNS, where one or more treatments are selected based on whether the mammal is identified as having an immune-mediated PNS as described herein (e.g., based, at least in part, on the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample obtained from a mammal). For example, a sample (e.g., a serum sample) obtained from a mammal (e.g., a mammal such as a human having a PNS) can be assessed for the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies), and one or more treatments can be selected and, optionally, administered, to the mammal based, at least in part, on whether the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) is detected.

In some cases, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) described herein can be used in an apheresis method to treat a mammal (e.g., a human) having a PNS. For example, a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) described herein can be used in an apheresis for the treatment of a PNS associated with anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) to remove anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) from the mammal. In some cases, an apheresis method to remove anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) from the blood of a mammal (e.g., a human such as a human having a PNS) can include withdrawing blood from the mammal; contacting the blood with a SK0R2 polypeptide and/or one or more fragments of a SK0R2 polypeptide (or a variant thereof) described herein that are, for example, immobilized to remove a substantial portion of anti- SK0R2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) from the blood; and returning the blood to the mammal. In some cases, methods and extracorporeal systems for apheresis (i.e., the process of withdrawing blood from an individual, removing components from the blood, and returning the blood, or blood depleted of one or more components, to the individual) can be used as described elsewhere (see, for example, U.S. Patent Nos. 4,708,713; 5,258,503; 5,386,734; and 6,409,696). As used herein, a “substantial portion” means removing at least 20% (e.g., at least 20%; 30%; 40%; 50%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 93%; 95%; 96%; 97%; 98%; 99%; 99.5%; 99.8%; or even 100%) of the anti-SK0R2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) that were present in the blood prior to removal.

When a mammal (e.g., a human) having a PNS is identified as having an immune- mediated PNS as described herein (e.g., based, at least in part, on the presence of anti- SK0R2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample obtained from a mammal), the mammal can be administered, or instructed to self-administer, one or more therapies effective to treat an immune-mediated PNS. In some cases, a therapy that can be effective to treat an immune-mediated PNS can include plasma exchange therapy (plasmapheresis). In some cases, a therapy that can be effective to treat an immune-mediated PNS can include intravenous immunoglobulin therapy. In some cases, a therapy that can be effective to treat an immune-mediated PNS can include administering one or more agents that can be effective to treat an immune-mediated PNS. Examples of agents that can be used to treat a mammal identified as having an immune-mediated PNS as described herein (e.g., based, at least in part, on the presence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS- specific autoantibodies) in a sample obtained from a mammal) include, without limitation, immunosuppressants (e.g., rituximab, mycophenolate, cyclophosphamide, and azathioprine), steroids (e.g., corticosteroids such as prednisone and methylprednisolone), anti-inflammatory agents, anti-seizure medications, and immunoglobulins (e.g., intravenous immunoglobulins).

When a mammal (e.g., a human) having a PNS is identified as having a PNS that is not immune-mediated (or is not associated with SKOR2-specific autoantibodies) as described herein (e.g., based, at least in part, on the absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample obtained from a mammal), the mammal can be administered, or instructed to self-administer, one or more therapies effective to treat a PNS that is not immune-mediated (e.g., one or more treatments that are not immunosuppressants). In some cases, a therapy that can be effective to treat a mammal identified as having a PNS that is not immune-mediated can include administering one or more agents that can be effective to treat a PNS that is not immune-mediated. Examples of agents that can be used to treat a mammal identified as having a PNS that is not immune- mediated (or is not associated with SKOR2-specific autoantibodies) as described herein (e.g., based, at least in part, on the absence of anti-SKOR2 polypeptide autoantibodies (e g., PNS- specific autoantibodies) in a sample obtained from a mammal) include, without limitation, anti-seizure medications and medications designed to enhance nerve-to-muscle transmission (e.g., pyridostigmine). Examples of therapies that can be used to treat a mammal identified as having a PNS that is not immune-mediated (or is not associated with SKOR2-specific autoantibodies) as described herein (e.g., based, at least in part, on the absence of anti- SK0R2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample obtained from a mammal) include, without limitation, physical therapy, speech therapy, cognitive behavioral therapy, and cognitive rehabilitation.

In some cases, when treating a mammal (e.g., a human) having a PNS (e.g., immune- mediated PNS or PNS that is not immune-mediated) as described herein, the treatment can be effective to reduce or eliminate one or more symptoms of the PNS. Examples of symptoms of a PNS include, without limitation, encephalitis, seizures, spastic ataxia, dysarthria, dysphagia, pseudobulbar affect, numbness, paraesthesia, pain, orthostatic intolerance, and incontinence (e.g., bowel incontinence and/or bladder incontinence). For example, the methods and materials described herein can be used to reduce one or more symptoms within a mammal having a PNS by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

In some cases, when treating a mammal (e.g., a human) having an immune-mediated PNS as described herein, the treatment can be effective to reduce or eliminate inflammation within the mammal (e.g., within one or more of the mammal’s muscles). For example, the methods and materials described herein can be used to reduce inflammation within a mammal having an immune-mediated PNS by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

In some cases, when treating a mammal (e.g., a human) an immune-mediated PNS as described herein, the treatment can be effective to reduce or eliminate the number of anti- SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) within the mammal. For example, the number of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) present within a mammal can be reduced using the methods and materials described herein. In some cases, the methods and materials described herein can be used to reduce the number of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) present within a mammal having an immune-mediated PNS by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the number of anti- SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) present within a mammal does not increase.

In some cases, methods for assessing a sample (e.g., a serum sample) obtained from a mammal (e.g., a human) having a PNS (e.g., immune-mediated PNS or PNS that is not immune-mediated) can be used to monitor a course of treatment. In some cases, a mammal being treated for a PNS and identified as having an immune-mediated PNS as described herein (e.g., based, at least in part, on the presence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) in a sample obtained from a mammal) can be monitored for the presence, absence, or level of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) to determine whether or not the immune-mediated PNS is being treated. For example, if the level of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) present within a mammal being treated as described herein is reduced following the administration of one or more immunosuppressants, the immune- mediated PNS is being treated. For example, if the level of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) present within a mammal being treated as described herein is not reduced following the administration of one or more immunosuppressants, the immune-mediated PNS is not being treated.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: SKOR2 IgG as a Biomarker of Paraneoplastic Neurologic Syndrome

This Example describes the identification of a Sloan Kettering virus family transcriptional corepressor 2 (SKOR2) polypeptide as the antigenic target for autoantibodies present in patients having a paraneoplastic neurologic syndrome (PNS). Methods

Patient selection

41 specimens (30 sera samples and 11 cerebrospinal fluids (CSFs)) that had a nuclear staining pattern at the junction of Purkinje cell layer and the granule layer of cerebellum when tested by an indirect immunofluorescence assay (IF A) on cryosectioned mouse tissues were collected. Of those, two had identical staining and were used for this project.

Phage immunoprecipitation sequencing (PhIP- Seq)

Sera from two patients with unique tissue immunofluorescence assay staining pattern were incubated with IO¹⁰ plaque-forming units per milliliter of the whole-human proteome phage-display library (Dubey et al., JAMA Neurol., 79(8):808-816 (2022)). The phage particles were incubated with serum (1 : 5000) or CSF (1 : 100) overnight at 4°C, isolated by magnetic protein G beads (Invitrogen), and extensively washed in immunoprecipitation wash buffer before the sequences of the bound phage were pulled out by polymerase chain reaction (PCR). These PCR sequences were then library prepped for next-generation sequencing using ILLUMINA® TruSeq™ Nano DNA library with associated indexes and sequenced with the ILLUMINA® NovaSeq™ platform. Sequenced reads were processed using an inhouse developed bioinformatics pipeline to identify the putative autoantigen.

Tissue immunofluorescence assay

Patient serum and commercial rabbit antibodies were tested on a cryosectioned composite of murine tissue (brain, kidney, and gut mucosa; Scimedx Corporation Denville, NJ). Sections were fixed using 4% paraformaldehyde for 1 minute, permeabilized with 3 -([3- cholamidopropyl] dimethylammonio)-l -propanesulfonate, 0.5%, in PBS for 1 minute, and then blocked for 1 hour with normal goat serum (10% in PBS). After a PBS rinse, sera and CSF patient specimen (serum preabsorbed at 1 :240 dilution with bovine liver powder; CSF nonabsorbed, 1 : 10 dilution) were applied and incubated for 40 minutes. This was followed by another PBS wash, and a 30-minute incubation with human IgG-specific secondary antibody (1 :2000) conjugated with FITC (Southern Biotechnology) in 10% goat serum PBS. After washing in PBS, cover slips were applied with ProLong Gold antifade mounting medium containing DAPI (Molecular Probes, Thermo Fisher Scientific). For dual staining on murine tissue, patient serum (1 :480) or CSF (1 :20) and rabbit polyclonal SKOR2-specific IgG (1 :300, Novus) and secondary antibodies (1 :200, tetramethylrhodamine-conjugated goat anti-rabbit IgG and goat anti-human IgG, Southern Biotechnology) were applied. Confocal images were captured using a LSM710 microscope (63 * or 40 or 20 x water immersion lens; Carl Zeiss Inc).

Cell-based assay

COS7 cells were transfected with plasma encoding full-length GFP tagged human SKOR2 (vector, pcDNA3.1 (+)-C eGFP plasmid, GenScript). Transfected COS7 cells were incubated for 16-24 hours (37°C, in humidified atmosphere of 95% air with 5% CO2). COS7 cells were then fixed (4% paraformaldehyde, 15 minutes), permeabilized (0.2% Triton X- 100, 10 minutes), and blocked for 1 hour with 10% normal goat serum in PBS. After a PBS wash, cells were incubated for 40 minutes with patient serum (1 :200 dilution), or rabbit SKOR2-specific IgG (1 :300). After PBS wash and incubation with secondary antibodies (1 :200 tetramethylrhodamine (TRITC)-conjugated goat antirabbit IgG and goat antihuman IgG; SouthemBiotech, Birmingham, AL). Coverslips were mounted using ProLong Gold antifade medium (containing 4,6-diamidino-2-phenylindole (DAPI); Molecular Probes, Thermo Fisher Scientific). Assays were scored by at least two independent reviewers.

Western blot

Forty hours following transfection with SKOR2-GFP plasmid, cells were washed once in cold IX PBS and one mF of cold RIPA (50 mM TrisHCL pH 7.5, 150 mM NaCL, 1.0% Triton-X, 0.1% SDS, Roche protease inhibitor tablet) was added. Cells were scrapped off the dish, lysates were homogenized and allowed to lyse in RIPA for an additional 30 minutes at 4°C before further homogenization. Lysate was used for western blotting (separated in a 4-15% polyacrylamide gel (Criterion Bio Rad) transblotted to nitrocellulose membranes (Life Science products) and blocked in 10% milk powder in tris-buffered saline and tween 20 (TBST). Individual lanes were probed with sera from healthy controls (1 : 100), candidate patients (1 : 100), or commercial SKOR2 specific IgG (1 : 1000). Nitrocellulose membrane was washed with TBST and probed with anti-human HRP or anti-rabbit HRP (Promega). Blots were visualized with SuperSignal West PicoPLus (thermo Scientific) and Azure Biosystems instrumentation.

Confirmation and verification of putative autoantigen

The putative autoantigen was confirmed by testing the two patients’ sera and/or CSF using a protein expression vector-transfected COS7 cell-based assay (CBA), western blotting, and IFA colocalization on cryosectioned mouse brain. A thorough clinical review of the two patients’ charts was performed.

In order to test the autoantigen specificity, serum samples from patients with adenocarcinoma without neurological syndromes (n=30), neuromyelitis optica spectrum disorder (NMOSD) (n=19), Sjogren’s syndrome (n=10), systemic lupus erythematosus (n=10), Purkinje cell cytoplasmic antibody type 1 (PCA1, anti-Yo) positives (n=6) and healthy controls (n=60), and CSF samples from multiple sclerosis (MS) (n=12) and normal pressure hydrocephalus (NPH) (n=10) were tested on CBA.

Additionally, tissue IFA specificity was evaluated by testing sera from patients with NMOSD (n=40), systemic lupus erythematosus (n=30), PCA1 IgG positives (n=9), antineuronal nuclear antibody type 1 (ANNA1, anti-Hu) IgG positives (n=20), and healthy controls (n=120), and CSF samples from multiple sclerosis (MS) (n=50) and normal pressure hydrocephalus (NPH) (n=25).

Immunohistochemistry

Formalin-fixed paraffin-embedded 5 pm thick sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed with EnVisionTM FLEX immunohistochemistry system (DAKO) after steam antigen retrieval with citric acid buffer (pH 6.0, DAKO). Primary antibodies against SKOR2 (1 :500, NBP2-12565, Novus biologicals) were incubated at 4°C overnight. Histopathology was reviewed and photographed with a brightfield microscope (Olympus BX53). Results

Discovery of the novel autoantigen

A unique nuclear murine tissue IF A staining pattern restricted to the junction of Purkinje cell layer and granule layer, with faint punctate staining in the brainstem and diencephalon was identified in samples of two patients (patient 1 had a serum titer > 1 : 1920 and a CSF titer > 1 : 100, and patient 2 had a serum titer > 1 : 1920; Figure 1 A). No staining was seen in the molecular layer of the cerebellum, hippocampus, cortex, gastric smooth muscle, gastric mucosa, or kidney. Serum and CSF from each of the two patients was tested by whole-human proteome PhlP-Seq. Bioinformatic analysis of the PhlP-Seq data revealed SKOR2 as the candidate antigen.

Validation of the novel autoantigen

Patient IgG from these specimens also colocalized with a commercial SKOR2- specific IgG on cryosections of mouse brain tissue by immunostaining (Figure IB). Both patient samples tested positive on CBA using SKOR2 -transfected COS7 cells (Figure 1C). Furthermore, patients’ sera yielded a positive band by western blot on lysate containing SKOR2 full length protein (Figure ID).

Specificity ofSKOR2 IgG

All healthy and disease control sera and/or CSF tested on IFA were negative. However, on CBA, one case of aquaporin-4 positive neuromyelitis optica was positive (all remaining NMOSD sera and other healthy/disease control sera and CSFs were negative on CBA). Consequently, this sample was tested on tissue indirect immunofluorescence assay which did not reveal the unique staining pattern or any colocalized with a commercial SKOR2-specific IgG. The sample was also tested by SKOR2 overexpression lysate western blot and it was found to be negative, suggesting a false positive SKOR2 CBA result.

Case presentations

SKOR2 IgG positive patients had subacute/chronic progressive central nervous system involvement, one presenting with encephalitis and seizures (case 1) and the other with spastic ataxia, dysarthria, dysphagia, cognitive dysfunction, and pseudobulbar affect (case 2).

Both patients were diagnosed of adenocarcinoma of the lung and gall bladder, respectively.

Case 1

A 64-year-old female was admitted to hospital after presenting with multiple episodes of right arm and leg sensorimotor focal seizures with impaired awareness progressing over the course of two weeks. On two occasions, these focal seizures progressed to bilateral tonic clonic seizures. She was noted to have a decline in cognition (specifically short-term memory) for six months prior (Figure 2A). MRI brain on admission showed T2/FLAIR hyperintensities in the left thalamus, external capsule, insula, and temporo-parietal cortex (Figure 2B1). Additionally, there were subtle T2/FLAIR hyperintensities in bilateral medial temporal lobes. Focal epileptiform discharges along with periodic lateralized discharges were seen in the left temporal lobe. There was also mild slowing in the same areas. CSF analysis revealed normal protein and cell count. No malignant cells were seen on CSF cytology. A 2.1 cm nodule in the upper lobe of left lung was seen on CT chest. Positron emission tomography (PET) scan of the whole body showed the nodule was intensely hypermetabolic and suggestive of malignancy (Figure 2B2), which on biopsy was confirmed to be invasive pulmonary adenocarcinoma. The tumor was considered inoperable due to poor patient status. Patient was initiated on radiation therapy (6000 cGy in eight fractions). The patient was initiated on four anti-seizure medications with limited improvement in clinical and electrographic seizures. For management of refractory seizures intravenous (IV) immunoglobulin (IG) 2 g/kg over 5 days was administered followed by IV methylprednisolone (IVMP) 1 gram daily for 5 days during the first 3 weeks of hospital admission. Subsequent imaging performed four weeks after treatment showed near complete resolution of T2/FLAIR hyperintensities, although the patient remained severely cognitively impaired (Figure 2B3). After being off immunotherapy for two months the patient had a clinical relapse in the form of worsening cognitive deficits and recurrence of seizures with MRI brain imaging showing recrudescence of previous findings (Figure 2B4). She was treated with cyclophosphamide (750 mg/m² monthly for 6 months - stopped after 2 treatments due to intolerability) following a repeat course of 1 g IVMP over 5 days, which contributed in some clinical improvement. She then received two induction doses of rituximab 1000 mg IV, separated by 14 days, approximately 6 months following her initial hospital admission. This was followed by two maintenance rituximab 500 mg IV treatments every six months following induction. Follow up MRI brain imaging 10 months from hospital admission showed mild residual signal abnormality of the left thalamus and insular cortex with development of global cerebral volume loss (Figure 2B5). Patient was found to have found have persistent severe cognitive impairment on the last clinical follow up (14 months post initial hospital admission, currently residing in long-term care facility).

Case 2

A 70-year-old right-handed female presented with complaints of gait imbalance (Figure 2C). A year after the onset of the gait dysfunction, she also developed dysarthria. Her examination revealed spastic dysarthria and pyramidal dysfunction in the 4 extremities, in the form of spasticity and hyperreflexia, in addition to bilateral Babinski sign. She had markedly slow alternating motion rate in tongue, hands, and legs. Her gait was wide based, ataxic, and spastic. Sensory examination was normal. MRI brain, cervical and thoracic spine were unremarkable. The electrodiagnostic studies revealed long duration high-amplitude polyphasic motor unit potentials, suggesting of a neurogenic process. No spontaneous insertional activity (fibrillations or fasciculations) was observed in any of the muscles tested. A comprehensive evaluation for myeloneuropathy etiologies including vitamin B12 level, folic acid level, human T-cell lymphotropic virus type 1/type 2 antibody, human immunodeficiency virus antigen, and genetic testing for spinocerebellar ataxias, Friedreich’s ataxia, and hereditary spastic paraplegia were negative. No cancer work up was performed during the initial evaluation. Over the next two years, she deteriorated and developed cognitive dysfunction, emotional lability along with dysphagia. She was subsequently found to have developed a widely metastatic gall bladder carcinoma which was not amenable to surgery and passed away.

SKOR2 tumor tissue immunoreactivity

Histopathological assessment of the lung adenocarcinoma tissue (case 1) from a single seropositive patient revealed cytoplasmic and nuclear SKOR2 immunoreactivity in the tumor cells (Figure 3). Together, these results demonstrate that a SK0R2 polypeptide is the antigenic target of autoantibodies present in PNS, and that a SK0R2 polypeptide fragment (e.g., a recombinant SK0R2 polypeptide fragment) can be used to detect the presence of autoantibodies present in PNS (e.g., anti-SKOR2 polypeptide antibodies) in samples (e.g., CSF samples).

Example 2: Identifying PNS

A blood sample (e.g., serum) is obtained from a human having a PNS. The obtained sample is contacted with a fragment of a SK0R2 polypeptide to form an antibody- SK0R2 fragment complex if the sample contains anti-SKOR2 polypeptide autoantibodies (e.g., PNS- specific autoantibodies), and then examined for the presence of an antibody- SK0R2 fragment complex.

If antibody- SK0R2 fragment complexes are detected in the sample, then the human is classified as having an immune-mediated PNS.

If antibody- SK0R2 fragment complexes are not detected in the sample, then the human is classified as having a PNS that is not immune-mediated (or is not associated with SKOR2-specific autoantibodies).

Example 3: Treating immune-mediated PNS

A sample (e.g., serum) is obtained from a human having a PNS. The sample is assessed for the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS- specific autoantibodies) by contacting the sample with a fragment of a SK0R2 polypeptide to form an antibody- SK0R2 fragment complex if the sample contains anti-SK0R2 polypeptide autoantibodies, and then examined for the presence of an antibody-SKOR2 fragment complex.

When the presence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) is detected in the sample, then the human is identified as having an immune- mediated PNS, and is administered one or more immunosuppressants (e.g., rituximab, mycophenolate, cyclophosphamide, and azathioprine) and/or one or more steroids (e.g., corticosteroids such as prednisone and methylprednisolone).

Example 4: Treating PNS

A sample (e.g., serum) is obtained from a human having a PNS. The sample is assessed for the presence or absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS- specific autoantibodies) by contacting the sample with a fragment of a SK0R2 polypeptide to form an antibody- SK0R2 fragment complex if the sample contains anti-SKOR2 polypeptide autoantibodies, and then examined for the presence of an antibody- SK0R2 fragment complex.

When the absence of anti-SKOR2 polypeptide autoantibodies (e.g., PNS-specific autoantibodies) is detected in the sample, then the human is identified as having a PNS that is not immune-mediated (or is not associated with SKOR2-specific autoantibodies), and is administered one or more anti-seizure medications and/or one or more medications designed to enhance nerve-to -muscle transmission (e.g., pyridostigmine).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for determining whether or not a mammal has an immune-mediated paraneoplastic neurologic syndrome (PNS), wherein said method comprises:

(a) contacting a sample from said mammal with a composition comprising an antigen to form an antigen-anti-Sloan Kettering virus family transcriptional corepressor 2 (SKOR2) polypeptide autoantibody complex if said sample comprises an anti-SKOR2 polypeptide autoantibody, wherein said antigen is a SKOR2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to said anti-SKOR2 polypeptide autoantibody, or a variant of said SK0R2 polypeptide having the ability to bind to said anti-SKOR2 polypeptide autoantibody; and

(b) detecting the presence or absence of said complex, wherein the presence of said complex indicates that said mammal has said immune-mediated PNS, and wherein the absence of said complex indicates that said mammal does not have said immune-mediated PNS.

2. The method claim 1, wherein said composition comprises a cell lysate obtained from a cell (a) comprising exogenous nucleic acid encoding said antigen and (b) expressing said antigen.

3. The method of any one of claims 1-2, wherein said antigen is said SK0R2 polypeptide.

4. The method of any one of claims 1-2, wherein said antigen is said variant.

5. The method of any one of claims 1-2, wherein said antigen is said fragment.

6. The method of claim 5, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO:2.

7. The method of any one of claims 1-6, wherein said antigen is covalently linked to a detectable label.

8. The method of claim 7, wherein said detectable label is selected from the group consisting of a green fluorescent protein (GFP) polypeptide, tetramethylrhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), a poly(His) tag, a glutathione-S- transferase (GST) tag, biotin, a Flag tag, and a myc tag.

9. The method of any one of claims 1-8, wherein said detecting comprises performing an immunological assay.

10. The method of any one of claims 1-9, wherein said mammal is a human.

11. The method of any one of claims 1-10, wherein said sample is a serum sample.

12. The method of any one of claims 1-11, wherein said method comprises detecting the presence of said complex.

13. The method of claim 12, wherein said method comprises classifying said mammal as having said immune-mediated PNS.

14. The method of any one of claims 1-11, wherein said method comprises detecting the absence of said complex.

15. The method of claim 14, wherein said method comprises classifying said mammal as not having said immune-mediated PNS.

16. The method of any one of claims 1-15, wherein said PNS is selected from the group consisting of cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonus- myoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, and dysautonomia.

17. A method for treating a mammal having an immune-mediated PNS, wherein said method comprises:

(a) determining that a sample from said mammal comprises the presence of anti- SKOR2 polypeptide autoantibodies, and

(b) administering an immunosuppressant to said mammal.

18. The method of claim 17, wherein said determining step comprises:

(i) contacting said sample with a composition comprising an antigen to form an antigen-anti-SKOR2 polypeptide autoantibody complex if said sample comprises an anti- SKOR2 polypeptide autoantibody, wherein said antigen is a SK0R2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to said anti-SKOR2 polypeptide autoantibody, or a variant of said SKOR2 polypeptide having the ability to bind to said anti- SK0R2 polypeptide autoantibody; and

(ii) detecting the presence of said complex, thereby determining that said mammal comprises the presence of said anti-SKOR2 polypeptide autoantibodies.

19. The method of claim 18, wherein said composition comprises a cell lysate obtained from a cell (a) comprising exogenous nucleic acid encoding said antigen and (b) expressing said antigen.

20. The method of any one of claims 18-19, wherein said antigen is said SKOR2 polypeptide.

21. The method of any one of claims 18-19, wherein said antigen is said variant.

22. The method of any one of claims 18-19, wherein said antigen is said fragment.

23. The method of claim 22, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO:2.

24. The method of any one of claims 18-23, wherein said antigen is covalently linked to a detectable label.

25. The method of claim 24, wherein said detectable label is selected from the group consisting of a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, and a myc tag.

26. The method of any one of claims 17-25, wherein said mammal is a human.

27. The method of any one of claims 17-26, wherein said sample is a serum sample.

28. The method of any one of claims 17-27, wherein said PNS is selected from the group consisting of cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonusmyoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, and dysautonomia.

29. The method of any one of claims 17-28, wherein said immunosuppressant is selected from the group consisting of rituximab, mycophenolate, cyclophosphamide, and azathioprine.

30. A method for treating an immune-mediated PNS, wherein said method comprises administering an immunosuppressant to a mammal having a PNS and that was identified as having anti-SKOR2 polypeptide autoantibodies.

31. The method of claim 30, wherein said identifying comprises:

(a) contacting a sample from said mammal with a composition comprising an antigen to form an antigen-anti- SKOR2 polypeptide autoantibody complex if said sample comprises an anti-SK0R2 polypeptide autoantibody, wherein said antigen is a SK0R2 polypeptide, a fragment of said SK0R2 polypeptide having the ability to bind to said anti-SK0R2 polypeptide autoantibody, or a variant of said SK0R2 polypeptide having the ability to bind to said anti-SK0R2 polypeptide autoantibody; and

(b) detecting the presence of said complex, thereby identifying said mammal as having said anti-SK0R2 polypeptide autoantibodies.

32. The method of claim 31, wherein said composition comprises a cell lysate obtained from a cell (a) comprising exogenous nucleic acid encoding said antigen and (b) expressing said antigen.

33. The method of any one of claims 31-32, wherein said antigen is said SK0R2 polypeptide.

34. The method of any one of claims 31-32, wherein said antigen is said variant.

35. The method of any one of claims 31-32, wherein said antigen is said fragment.

36. The method of claim 35, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO:2.

37. The method of any one of claims 31-36, wherein said antigen is covalently linked to a detectable label.

38. The method of claim 37, wherein said detectable label is selected from the group consisting of a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, and a myc tag.

39. The method of any one of claims 30-38, wherein said mammal is a human.

40. The method of any one of claims 30-39, wherein said sample is a serum sample.

41. The method of any one of claims 30-40, wherein said PNS is selected from the group consisting of cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonusmyoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, and dysautonomia.

42. The method of any one of claims 30-41, wherein said immunosuppressant is selected from the group consisting of rituximab, mycophenolate, cyclophosphamide, and azathioprine.

43. A method for treating a mammal having a PNS, wherein said method comprises:

(a) determining that a sample from said mammal lacks the presence of anti-SKOR2 polypeptide autoantibodies, and

(b) administering a therapy for said PNS to said mammal, wherein said therapy is not an immunosuppressant.

44. The method of claim 43, wherein said determining step comprises:

(i) contacting said sample with a composition comprising an antigen to form an antigen-anti-SKOR2 polypeptide autoantibody complex if said sample comprises an anti- SKOR2 polypeptide autoantibody, wherein said antigen is a SKOR2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to said anti-SKOR2 polypeptide autoantibody, or a variant of said SKOR2 polypeptide having the ability to bind to said anti- SKOR2 polypeptide autoantibody; and

(ii) detecting the absence of said complex, thereby that said mammal lacks the presence of anti-SKOR2 polypeptide autoantibodies.

45. The method of claim 44, wherein said composition comprises a cell lysate obtained from a cell (a) comprising exogenous nucleic acid encoding said antigen and (b) expressing said antigen.

46. The method of any one of claims 44-45, wherein said antigen is said SKOR2 polypeptide.

47. The method of any one of claims 44-45, wherein said antigen is said variant.

48. The method of any one of claims 44-45, wherein said antigen is said fragment.

49. The method of claim 48, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO:2.

50. The method of any one of claims 44-49, wherein said antigen is covalently linked to a detectable label.

51. The method of claim 50, wherein said detectable label is selected from the group consisting of a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, and a myc tag.

52. The method of any one of claims 43-51, wherein said mammal is a human.

53. The method of any one of claims 43-52, wherein said sample is a serum sample.

54. The method of any one of claims 43-53, wherein said PNS is selected from the group consisting of cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonusmyoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, and dysautonomia.

55. The method of any one of claims 43-54, wherein said therapy for said PNS is selected from the group consisting of a physical therapy, speech therapy, cognitive behavioral therapy, and cognitive rehabilitation.

54. A method for treating a PNS, wherein said method comprises administering a therapy for said PNS to a mammal that was identified as lacking anti-SKOR2 polypeptide autoantibodies, wherein said therapy is not an immunosuppressant.

55. The method of claim 54, wherein said identifying comprises:

(a) contacting a sample from said mammal with a composition comprising an antigen to form an antigen-anti- SK0R2 polypeptide autoantibody complex if said sample comprises an anti-SKOR2 polypeptide autoantibody, wherein said antigen is a SKOR2 polypeptide, a fragment of said SK0R2 polypeptide having the ability to bind to said anti-SKOR2 polypeptide autoantibody, or a variant of said SK0R2 polypeptide having the ability to bind to said anti-SKOR2 polypeptide autoantibody; and

(b) detecting the absence of said complex, thereby identifying said mammal as lacking anti-SKOR2 polypeptide autoantibodies.

56. The method claim 55, wherein said composition comprises a cell lysate obtained from a cell (a) comprising exogenous nucleic acid encoding said antigen and (b) expressing said antigen.

57. The method of any one of claims 55-56, wherein said antigen is said SK0R2 polypeptide.

58. The method of any one of claims 55-56, wherein said antigen is said variant.

59. The method of any one of claims 55-56, wherein said antigen is said fragment.

60. The method of claim 59, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO:2.

61. The method of any one of claims 55-60, wherein said antigen is covalently linked to a detectable label.

62. The method of claim 61, wherein said detectable label is selected from the group consisting of a GFP polypeptide, TRITC, FITC, a poly(His) tag, a GST tag, biotin, a Flag tag, and a myc tag.

63. The method of any one of claims 54-62, wherein said mammal is a human.

64. The method of any one of claims 54-63, wherein said sample is a serum sample.

65. The method of any one of claims 54-64, wherein said PNS is selected from the group consisting of cerebellar ataxia, limbic encephalitis, encephalomyelitis, opsoclonusmyoclonus, stiff person syndrome, myelopathy, lambert-eaton myasthenic syndrome, myasthenia gravis, neuromyotonia, peripheral neuropathy, and dysautonomia.

66. The method of any one of claims 54-65, wherein said therapy for said PNS is selected from the group consisting of a physical therapy, speech therapy, cognitive behavioral therapy, and cognitive rehabilitation.

67. A method for treating an immune-mediated PNS, wherein said method comprises:

(a) removing blood from a mammal having said immune-mediated PNS, thereby obtaining removed blood,

(b) removing at least some anti-SKOR2 polypeptide autoantibodies from said removed blood, thereby obtaining processed blood, and

(c) reintroducing said processed blood into said mammal.

68. The method of claim 67, wherein said step (b) comprises contacting said removed blood with immobilized antigen, wherein said antigen is a SKOR2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, or a variant of said SK0R2 polypeptide having the ability to bind to an anti- SK0R2 polypeptide autoantibody.

69. The method of claim 68, wherein said antigen is said SK0R2 polypeptide.

70. The method of claim 68, wherein said antigen is said variant.

71. The method of claim 68, wherein said antigen is said fragment.

72. The method of claim 71, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO: 2.

73. The method of any one of claims 68-72, wherein said mammal is a human.

74. A method for treating a PNS, wherein said method comprises:

(a) removing blood from a mammal having said PNS that was identified as having anti-SKOR2 polypeptide autoantibodies, thereby obtaining removed blood,

(c) reintroducing said processed blood into said mammal.

75. The method of claim 74, wherein said step (b) comprises contacting said removed blood with immobilized antigen, wherein said antigen is a SKOR2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, or a variant of said SKOR2 polypeptide having the ability to bind to an anti- SKOR2 polypeptide autoantibody.

76. The method of claim 75, wherein said antigen is said SKOR2 polypeptide.

77. The method of claim 75, wherein said antigen is said variant.

78. The method of claim 75, wherein said antigen is said fragment.

79. The method of claim 78, wherein said fragment consists of the amino acid sequence set forth in SEQ ID NO: 2.

80. The use of a composition comprising an antigen to treat an immune-mediated PNS, wherein said antigen is a SKOR2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, or a variant of said SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody.

81. The use of a composition comprising an antigen to treat an immune-mediated PNS, wherein said antigen is a SKOR2 polypeptide, a fragment of said SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, or a variant of said SKOR2 polypeptide having the ability to bind to an anti-SKOR2 polypeptide autoantibody, wherein said use comprises removing at least some anti-SKOR2 polypeptide autoantibodies from the blood of a mammal having said immune-mediated PNS.