# Patent

## Title
Assay for anti-TR antibodies

## Abstract

The present invention relates to a method for determining the presence of anti-Tr antibodies in a subject comprising the steps of obtaining a sample from said subject testing the presence of said antibodies in said sample by addition of DNER protein or an antigenic part thereof and checking whether said DNER protein is bound by any antibodies in said sample Such an assay is useful for the diagnosis of paraneoplastic cerebellar degeneration that is associated with Hodgkin lymphoma, or, more generally, to type patients suffering from cerebellar ataxia. Also comprised in the invention is a kit for performing such an assay.

## Background
FIELD OF THE INVENTION
The invention relates to the field of immunoassays, more particularly to the filed of assays in neurological diseases, more specifically with relation to paraneoplastic cerebellar degeneration.
INTRODUCTION
Paraneoplastic cerebellar degeneration (PCD) is a heterogenous group of neurological disorders characterized by subacute cerebellar ataxia which coincides with the presence of specific tumour types and antineural antibodies.
The clinical cerebellar ataxia symptoms that are evident in patients with PCD are caused by Purkinje neuronal loss or by loss of function of Purkinje neuronal cells in the cerebellum; it is manifested by dysarthria, limb and gait ataxia, and nystagmus. Radiologic imaging occasionally reveals cerebellar atrophy.
As is the case with other paraneoplastic syndromes, PCD is believed to be due to an autoimmune reaction targeted against components of the central nervous system (in PCD, this is specifically Purkinje cells). It is thought to be triggered when tumor cells (in PCD, most commonly ovarian or breast cancer) express a protein normally expressed in the brain (in PCD, this is the Purkinje neuronal protein termed cdr2). This is believed to trigger an anti-tumor immune response that may be clinically significant, but also an anti-neuronal immune response. Such PCD patients harbor an anti-neuronal antibody known as anti-Yo (named after the first two letters of the index patient). PCD may be associated with other tumors—when associated with small cell lung cancer, it is associated with an antibody termed “anti-Hu” (more commonly associated with paraneoplastic subacute sensory neuropathy and/or limbic encephalitis), but also further antibodies (anti-Ri, anti-Ma, anti-CV2, anti-amphiphysin) have been identified.
One particular form of PCD is associated with Hodgkin lymphoma (HL) and is characterized by the presence of anti-Tr antibodies. These antibodies recognize a specific punctuate immunoreactivity in the large dendritic tree as well as in the soma of Purkinje cells, but not in the axons. Although the disease itself was recognised already long ago (Horwich, L. et al., 1966, J. Neurol. Neurosurg. Psychiatry 29:45-51), the detection of the anti-Tr antibodies in 1997 (Graus, F. et al., 1997, J. Neuroimmunol. 74:55-61) showed that these were associated with the syndrome. Since then, it has been searched for the antigen to which these antibodies are directed. However, until now such an antigen, despite all the research involved, has not been identified.
Diagnosis of this particular form of Hodgkin lymphoma associated PCD is seriously hampered by the fact that no antigen is available. Now, only an indirect method to show the presence of the anti-Tr antibody is used. Such a test is currently offered commercially (e.g. by Immco Diagnostics). The characteristic punctuate staining of anti-Tr sera is only one prerequisite of the diagnosis: the diagnosis is usually confirmed by performing antigen blocking experiments (Shams'ili S. et al., 2003, Brain 126:1409-1418; Bernal, F. et al., 2003, Neurology 53:1719-1723). For this, cerebellar sections are first pre-incubated with undiluted anti-Tr sera that require testing and subsequently incubated with diluted biotinylated standard anti-Tr serum. This method, by definition, is less reliable than detection by an assay with the antigen itself. Thus, there is ample need for identification of the antigen and incorporating this into an assay for the detection of the presence of the anti-Tr antibody and, because of that, diagnosis of the presence of HL-associated PCD.
SUMMARY OF THE INVENTION
The current inventors have been able to identify the antigen that is capable of binding to the anti-Tr auto-antibody.
The invention therefore comprises a method for determining the presence of anti-Tr antibodies in a subject comprising the steps of

    
    
        a. obtaining a sample from said subject
        b. testing the presence of said antibodies in said sample by addition of DNER protein or an antigenic part thereof and checking whether said DNER protein is bound by any antibodies in said sample.
    
    


Preferably in said method the DNER protein or antigenic part thereof is labeled. In a further preferred embodiment the sample is a blood, plasma, serum or CSF sample.
Such a method preferably entails an immunoassay, more preferably a cell-based immunoassay.
In a further preferred embodiment the method is applied to a human subject, preferably wherein the human subject is suffering from paraneoplastic cerebellar degeneration and/or ataxia.
Alternatively, the invention comprises a method for detection of Hodgkin lymphoma by performing a method according to the invention.
Further comprised in the present invention is a method for typing subjects suffering from paraneoplastic cerebellar degeneration and/or ataxia comprising performing a method according to the invention.
Also part of the invention is the use of the DNER protein or an antigenic part thereof for the detection of anti-Tr antibodies. Preferably in such a use the DNER protein or an antigenic part thereof is labeled.
Alternatively, the invention comprises the use of a cell line transfected with a construct encoding a DNER protein or antigenic part thereof for the detection of anti-Tr antibodies.
In another aspect the invention comprises a kit for performing an immunoassay wherein said kit comprises a DNER protein or antigenic part thereof and/or a cell line that is capable of producing said protein or part thereof, and an anti-Tr antibody as positive control. Preferably in such a kit the DNER protein or antigenic part thereof is labeled.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-D show identification of DNER as the Tr antigen.
FIGS. 2A-H show immunohistochemistry of anti-DNER and anti-Tr sera on rat cerebella.
FIGS. 3A-B show knock down of DNER abolishes Tr reactivity in hippocampal neurons.
FIGS. 4A-E show Tr epitope mapping of DNER.
FIG. 5 shows DNER sequence (SEQ. ID NO. 24).



FIG. 1. IDENTIFICATION OF DNER AS THE TR ANTIGEN
A) Coomassie stained gel of total rat brain extracts immunoprecipitated with anti-Tr positive sera and used for mass spectrometry analysis.
B) HeLa cells expressing HA-DNER (green) are positively stained by two anti-Tr sera (Tr9 and Tr6, 1:200, red), CSF of the same patient (Tr6, 1:100, red) but not by the control sera (ctrl, 1:200, red). The merged images reveal non-transfected cells by DAPI staining. Scale bar: 10 μm.
C) Western blot analysis of total HEK293T cell extracts expressing HA-DNER (input) and purified HA-DNER (HA-DNER). Untransfected HEK293T cells (mock) are used as control. HA-DNER was purified by immunoprecipitation using anti-HA antibodies. The asterisk's denote background bands also present in untransfected HEK293T cells.
D) Representative images of hippocampal neurons (days in vitro 11) stained with anti-DNER (green) and anti-Tr serum Tr9 (red). Zoom indicates punctuate DNER staining in neurites that strongly co-localize with anti-Tr serum. Scale bar: 20 μm.
FIG. 2. IMMUNOHISTOCHEMISTRY OF ANTI-DNER AND ANTI-TR SERA ON RAT CEREBELLA
(A-B) Sagittal section of rat brain stained with serum of a patient known to contain the anti-Tr antibodies (A) or commercial anti-DNER (B). A′-B′: Higher magnification shows that anti-DNER antibodies lead to the identical dot-like somatodendritic staining as described for anti-Tr sera.
(C-E) Standard blocking procedure currently used to test for presence of anti-Tr antibodies in sera. (C) Punctate staining after incubation with standard biotinylated serum. (D-E) Staining disappears when previously incubated with unbiotinylated serum from the same patient (D) or serum from another patient (E).
(F-H) Depletion of anti-DNER antibodies from anti-Tr positive sera (F) obliterates the anti-Tr staining pattern (G). The eluted fraction, containing purified anti-DNER antibodies, retains the normal punctuate staining (H). Note that a lower dilution was used in F-H (1/20.000) than in B-E (1/400). Scale bar: 200 μm in A, B and 25 μm in A′-B′, C-D, F-H.
FIG. 3. KNOCK DOWN OF DNER ABOLISHES TR REACTIVITY IN HIPPOCAMPAL NEURONS
A) In neurons transfected with a control construct, normal DNER (green) and Tr (red) reactivity is observed (upper panel). In all neurons transfected with shRNA constructs expressing shRNA against DNER and β-Galactosidase (blue) both DNER expression and Tr reactivity is lacking (lower panel). Scale bar: 20 μm. B) Summary of DNER knock down experiments. Quantification of the number of TR and DNER positive neurons expressing control shRNA (n=162), DNER-shRNA#1 (n=133) or DNER-shRNA#2 (n=134).
FIG. 4. TR EPITOPE MAPPING OF DNER
(A) The upper panel gives a schematic representation of DNER to indicate the epitope regions (numbers depict amino acid, light grey boxes indicate EGF domains, SP: N-terminal signal peptide, TM: transmembrane domain). Lower panel: Epitope mapping of DNER by expressing HA- or myc-tagged DNER deletion constructs (green) in HeLa cells stained by anti-Tr sera (red) displays two groups of patients: Tr9 detects one epitope encompassing aa 128-308 only, whereas Tr8 detects a second epitope within aa 302-675. Scale bar, 10 μm.
(B) Summary of the cell-based Tr epitope mapping experiments with the 13 different anti-Tr sera, including the Tr-positive serum from the HL patient without ataxia.
(C) Western blot analysis reveals that Tunicamycin treatment of HA-DNER expressing HEK293T cells inhibits the glycosylation of DNER as seen by the reduction in molecular weight. The anti-Tr positive sera (Tr9 and Tr5) no longer recognize unglycosylated DNER.
(D) Mutating four N-glycosylation sites in DNER1-737 abolishes the recognition by the anti-Tr sera (Tr9 and Tr10) as analyzed on Western blotting.
(E) Mutating four N-glycosylation sites in DNER128-308 completely abolishes the recognition by 9 anti-Tr sera, whereas the remaining 3 sera only gave minimal reactivity (see also B).
FIG. 5. DNER SEQUENCE
The protein has a large extracellular domain, ranging from amino acid 35-640 (the first 34 amino acids form the signal peptide). The transmembrane part only comprises 20 amino acids (641-661), while the intracellular part forms the C-terminal end of the molecule (amino acids 662-735). The extracellular domain contains 10 EGF like domains:
EGF like 1 domain 44-92
EGF like 2 domain 94-133
EGF like 3 domain 309-348
EGF like 4 domain 349-390
EGF like 5 domain 392-428
EGF like 6 domain 430-466
EGF like 7 domain 468-503
EGF like 8 domain 505-541 (potential calcium binding)
EGF like 9 domain 543-579
EGF like 10 domain 581-617 (potential calcium binding)
EGF like 1 and 2 taken together are thought—on basis of sequence similarity—to interact with NOTCH1.
Glycosylation takes place on amino acid 223 and 564.
DETAILED DESCRIPTION OF THE INVENTION
In recent years, progress has been made in the identification of various antineuronal antibodies in different ways. Overlapping expression patterns led to the identification of antibodies against the metabotropic glutamate receptor 1 (mGluR1) in PCD associated with HL (Sillevis Smitt, P. et al., 2000, N. Engl. J. Med. 342:21-27) and anti-N-methyl-D-aspartate receptor (NMDAR) antibodies in limbic encephalitis (Dalmau J. et al., 2007, Ann. Neurol. 61:25-36) while association with a known complex identified Leucine-rich, glioma inactivated 1 (Lgi-1) as the antigen recognized by antibodies against voltage gated potassium channels (VGKC) (Irani, S. R. et al., 2010, Brain 133:2734-2748; Lai M., et al., 2010, Lancet Neurol. 9:776-785). Recently immunoprecipitation followed by mass spectrometry analysis (IP-MS) directed the identification of antibodies against the glutamate receptor subunits GluR1 and GluR2 (Lai M. et al., 2009, Ann. Neurol. 65:424-434), the Gamma Amino Butyric Acid B (GABAB) Receptor-1 and -2 subunits (Lancaster E. et al., 2010, Lancet Neurol. 9:67-76) and mGluR5 (Lancaster E. et al., 2011, Neurology 77:1698-1701) in limbic encephalitis. In these cases, clear bands were already visible on coomassie stained gels after immunoprecipitation. This was not the case with the anti-Tr antibodies that are associated with HL in PCD. In order to proceed we took advantage of the fact that all anti-Tr sera recognize the same epitope (FIG. 2C-E and Bernal et al 2003). This meant that we had to analyze the entire peptide data set obtained by IP-MS in order to identify proteins that were precipitated by all 4 anti-Tr sera.
In the end, however, the present inventors were able to conclude that the protein DNER (Delta/Notch-like Epidermal growth factor related Receptor) contained the epitope that caused the binding of the anti-Tr antibodies and thus is the long-sought antigen. As is shown in the experimental part, the fact that DNER is the antigen has been confirmed by histochemical evidence. This finding enables the production of a quick and reliable assay for screening for anti-Tr antibodies. We mapped the main epitope for all 13 anti-Tr sera (including the serum from a HL patient without ataxia) to an extracellular region of 176 amino acids between amino acids 128-304 of DNER. It further appeared that that Tr antigenicity is dependent on N-glycosylation of this region. Whether this is caused by recognition of the glycogen chains or due to the folding of the protein remains to be determined and has no implications on the current invention. Final proof was given by the fact that anti-Tr sera depleted of DNER-antibodies no longer displayed the typical somatodendritic punctuate staining. Although the main epitope recognized by all 12 anti-Tr positive sera from PCD patients was mapped to an extracellular 176 aa region between EGF domains 2 and 3, five of these sera could recognize a second epitope, also in the extracellular region of DNER. The clinical phenotype, known in 4 of these 5 patients, was not different from the seven other patients (Table 1). The 13th anti-Tr positive serum from the non-ataxic HL patient also detected both epitopes, identical to the five PCD patients.
The Tr antigen DNER is expressed throughout the brain during development and adulthood, but is highly expressed in the Purkinje cells in a punctuate somatodendritic manner (Eiraku, M. et al., 2002, J. Biol. Chem. 277:25400-25407; Nishizumi, H. et al., 2002, Neuroreport. 13:909-915). Similar to the subacute cerebellar ataxia observed in anti-Tr positive patients, mice lacking DNER exhibited motor discoordination in the fixed bar and rotarod tests. Moreover, the cerebellum from knockout mice showed significant retardation in morphogenesis and persistent abnormality in fissure organization (Tohgo, A. et al., 2006, Mol. Cell Neurosci. 31:326-333).
DNER protein is an epidermal growth factor (EGF)-like repeat-containing single-pass transmembrane protein that is specifically expressed in the developing and mature central nervous system. Sequence analysis shows that the 10 EGF-like repeats in the extracellular domain are closely related to those of the developmentally important receptor Notch and its ligand Delta. Accordingly the protein was named Delta/Notch-like EGF-related receptor (DNER). DNER protein is strongly expressed in several types of post-mitotic neurons, including cortical and hippocampal pyramidal neurons, cerebellar granule cells, and Purkinje cells. DNER protein is localized to the dendritic plasma membrane and endosomes and is excluded from the axons, even when overexpressed.
The identification of DNER as Tr antigen in this study simplifies the method for determining the presence of anti-Tr antibodies in patients with subacute cerebellar symptoms. Instead of using the strict immunohistochemical criteria followed by a blocking experiment (Bernal et al., supra), a simple assay based on the expression of exogenous DNER can now be used.
Accordingly, the present invention is directed to an immunoassay method for typing patients with paraneoplastic cerebellar degeneration and/or cerebellar ataxia comprising testing for the presence of anti-Tr antibodies with its antigen DNER or an antigenic part thereof. For this invention an antigenic part of the DNER protein is defined as that part of the DNER protein that is capable of binding to the anti-Tr antibody. Thus an antigenic part of the DNER protein can for example be the part between EGF-like domain 2 and 3.
In principle the test can be any sort of (immuno-)assay in which the binding between the DNER protein (or antigenic part thereof) with the anti-Tr antibody which is to be detected can be established. Preferably, to enable detection, the DNER protein or antigenic part thereof is labeled, for which labeling any suitable label can be used. Suitable labels for instance could be chosen from enzymes, dyes, radioactive moieties, specific tags that can be recognised by secondary antibodies, fluorescent proteins (e.g. GFP) etc.
For such a test a sample taken from a subject that is suspected of having PCD with HL is used, which sample is a sample from a body liquid, preferably is a blood sample (like a serum or plasma sample) or a cerebrospinal fluid (CSF) sample.
The subject preferably is a human.
Thus, the test can be a RIA (radio-immunoassay), EIA (enzyme immunoassay), an ELISA (enzyme-linked immuno-sorbent assay) or any other type of immunoassay and such an assay may be direct, sandwich, competitive or non-competitive or any other type of assay. The assay may involve solid supports such as beads, columns, (glass or ceramic) plates and the like and it may be conducted on micro-titer plates, reagent cups or any suitable holder.
Preferably, the assay is a cell-based assay in which the labeled antigen is (recombinantly) produced by a cell. Such a cell is preferably a mammalian and more preferably a human cell, such as a CHO cell, a HeLa cell, a HEK-293 cell, a Vero cell, a 293-T cell, a 3T3 cell, a H1299 cell, a Jurkat cell, a JY cell, or a Raji cell. Cells can be transiently or stably transfected with a construct that enables expression of the DNER protein or an antigenic part thereof. Production of such a construct that enables expression of the DNER protein or an antigenic part thereof, transfection of a cell with such a construct, culturing said cell and harvesting the cultured cells or the produced DNER protein is well within the capability of the skilled person. Further, in the experimental part a clear description is given of an example how to perform an assay as claimed.
The assay is preferably used when a subject is suspected of suffering from PCD with HL, but it can also be a part of a series of assays to type subjects suffering from PCD or cerebellar ataxia. In such a case, also assays for the detection of anti-Yo, anti-Hu, anti-Ri, anti-Ma, anti-CV2, anti-mGluR1 and/or anti-amphiphysin antibodies maybe comprised in the series of assays. The term “typing of subjects” in this application therefore means to detect which anti-neuronal antibodies (and/or which tumours) are present in the subject and are responsible for the clinical signs of PCD or cerebellar ataxia. Thus also a method for the typing of subjects using an assay according to the invention is part of the current invention.
Further part of the invention is a kit for performing the assays as described. Such a kit will comprise the DNER protein or an antigenic part thereof, and/or a cell line that can produce said protein or part thereof. Further, the kit will preferably comprise an anti-Tr antibody to be used as a positive control. Also instructions for use of the kit may be present. If the assay is used as part of a typing protocol for PCD patients, the kit may further comprise additional assay requirements for testing of anti-neuronal antibodies such as anti-Yo, anti-Hu, anti-Ri, anti-Ma, anti-CV2, anti-mGluR1 and/or anti-amphiphysin antibodies.
Experimental Part
Patients
Twelve anti-Tr positive patients were selected in our hospital. The most relevant clinical features of 11 of the 12 anti-Tr positive patients are given in Table 1. Clinical information was lacking from Tr-positive patient 5. All patients suffered subacute and severe truncal and limb ataxia with nystagmus and/or cerebellar dysarthria. Ten patients were diagnosed with HL and in all of them the cerebellar symptoms preceded the detection of HL.
Assessment of Patient's Disability
The patients' disability was assessed using a modified Rankin scale (mRS) (Keime-Guibert, F. et al., 1999, Neurology 53:1719-1723). On the mRS a score of 0 represents an asymptomatic patient; 1, symptoms that do not interfere with lifestyle; 2, symptoms that lead to some restriction of lifestyle but do not prevent totally independent existence; 3, symptoms significantly interfere with lifestyle or prevent totally independent existence; 4, symptoms clearly prevent independent existence, although the patient does not need constant attention; 5, severe disability with total dependence requiring constant attention; 6, death from neurological cause 4.
Antibodies and Expression Constructs
For immunocytochemistry, antibodies were used in the following dilutions: mouse anti-β-galactosidase (Promega, Leiden, Netherlands) 1:2,000, goat anti-DNER (R&D systems, Abingdon, United Kingdom) 1:400-1:2,000, rabbit anti-HA (Santa-CruzHeidelberg, Germany) 1:400, patients' sera: 1:50-1:20,000. Cy3-conjugated donkey anti-Human (Jackson's lab, Newmarket, UK), Alexa488- and Alexa568-conjugated secondary antibodies (Molecular Probes, Breda, Netherlands) and FITC-conjugated anti-goat (Jackson) were used in a 1:400 dilution. For Western blotting: HRP-conjugated donkey anti-human (Calbiochem, Darmstadt, Germany) 1:10,000 and HRP-conjugated swine anti-rabbit, -mouse or -goat (Dako, Heverlee, Belgium) 1:2500 were used.
Full length wild type HA-tagged mouse DNER cDNA in pDisplay was kindly provided by M. Kengaku (Kyoto) and was previously described10. DNER-GFP-bio, the deletion and glycosylation mutants were generated by PCR and cloning in pGW111.
Primers Used:














SEQ




ID


Name
sequence (5′-3′)
NO

















dnerF1
Ccagtgtgctggaattcg
1


 


dnerR1
ctgGTCGACaaatcttttgttttaatcag
2


 


dnerΔ1F
cggctatgaaggtcttaactgtgctagcaactgtg
3



ttccgg



 


dnerΔ1R
ccggaacacagttgctagcacagttaagaccttca
4



tagccg



 


dnerR2
ctgGTCGACcaaatcttttgttttaatcag
5


 


dnerE2R
ctggtcgactggttgttcacagttaagacc
6


 


dnerE2F
cctgaattcggtcttaactgtgaacaacc
7


 


dnerE3R
caggtcgacggaacacagttgctagcc
8


 


dnerE3F
caggaattcgctagcaactgtgttccg
9


 


dnerTMR
gcagtcgactatggccgggaggagccc
10


 


delG1F
gatgttgcctgtgggGCtgcTagCtccaacaac
11



tctgcg



 


delG1R
cgcagagttgttggaGctAgcaGCcccacaggc
12



aacatc



 


delG2F
gccagttccGCcaactctgcgggtgg
13


 


delG2R
ccacccgcagagttgGCggaactggc
14


 


delG3F
gaagtgccacagGCcactAGTgtaaagattcgg
15



cagg



 


delG3R
cctgccgaatctttacACTagtgGCctgtggca
16



cttc



 


delG4F
cttcattggttttgtgGCtgactctgttgc
17


 


delG4R
gcaacagagtcaGCcacaaaaccaatgaag
18









Constructs:













HA-DNER1-737
PCR:dnerF1/dnerR1, EcoRI-SalI into pGW1


DNER GFPbio
PCR:dnerF1/dnerR2, EcoRI-SalI into



pGW1-GFPbio


HA-DNER1-134
PCR:dnerF1/dnerE2R, EcoRI-SalI into pGW1


myc-DNER128-308
PCR:dnerE2F/dnerE3R, EcoRI-SalI into pGW1,



insert recloned into AscI-Noti GW-Spmyc


myc-DNER304-675
PCR:dnerE3F/dnerTMR, EcoRI-SalI into pGW1,



insert recloned into AscI-NotI pGW-Spmyc


HA-DNERΔ128-304
PCRdnerF1/dnerΔ1F/dnerΔ1R/dnerR1,



EcoRI-SalI into pGW1


HA-DNERΔG1-4
consecutively: delG1,2,3,4F/R&dnerF1/dnerR1,



EcoRI-SalIinto pGW1









The template for all constructs was the HA-DNER in pDISPLAY, kindly provided by M. Kengaku1. All DNER constructs were subcloned in pGW1 expression vectors for optimal neuronal expression2. A linker encoding the sequence ASGLNDIFEAQKIEWHEGGG, which is the substrate of biotin ligase BirA was inserted into the AscI EcoRI sites after the GFP in pGW1-GFP3. In pGW-SPmyc a linker encoding the signal peptide MNFIPVDIPLLMIFLVTTGGSALEKLAT was inserted before the myc-tag MEGKLISEEDL.
Immunoprecipitation and Mass Spectrometry
Extracts were made from adult total rat brain using 1% NP40 lysis buffer (10 mM Hepes, pH 7.5, 150 mM NaCl, 1% NP40 and complete protease inhibitor (Roche). 2.5 mg protein was precleared with 50 ul protA/protG sepharose, mixed 1:1, for 1 hour at 4° C. and the supernatant was subsequently incubated overnight at 4° C. with 10 ul sera. The following morning 50 ul protA/protG (1:1) sepharose was added and left to incubate for an additional 4 hours at 4° C. The sepharose beads were washed 5 times with lysis buffer and loaded on a 4-12% Bis-Tris gel (Invitrogen, Breda, Netherlands).
Mass spectrometry was mainly performed as described before (Lansbergen, G. et al., 2006, Dev. Cell. 11:21-32; Jaworski, J. et al., 2009, Neuron. 61: 85-100). For mass spectrometry analysis, 1D SDS-PAGE gel lanes were cut into 2-mm slices using an automatic gel slicer and subjected to in gel reduction with dithiothreitol, alkylation with iodoacetamide and digestion with trypsin (sequencing grade, Promega, Leiden, Netherlands), essentially as described previously (Wilm, M. et al., 1996, Nature 379:466-469). Nanoflow LCMS/MS was performed on an 1100 series capillary LC system (Agilent Technologies, Amstelveen, Netherlands) coupled to an LTQ linear ion trap mass spectrometer (Thermo, Breda, Netherlands) operating in positive mode and equipped with a nanospray source. Peptide mixtures were trapped on a ReproSil C18 reversed phase column (column dimensions 1.5 cm×100 μm, packed in-house; Dr Maisch GmbH, Ammerbuch-Entringen, Germany) at a flow rate of 8 μl/min. Peptide separation was performed on ReproSil C18 reversed phase column (column dimensions 15 cm×50 μm, packed inhouse; Dr Maisch GmbH, Ammerbuch-Entringen, Germany) using a linear gradient from 0 to 80% B (A=0.1 M acetic acid; B=80% (v/v) acetonitrile, 0.1 M acetic acid) in 70 min and at a constant flow rate of 200 nl/min using a splitter. The column eluent was directly sprayed into the ESI source of the mass spectrometer. Mass spectra were acquired in continuum mode; fragmentation of the 4 peptides was performed in data-dependent mode. Peak lists were automatically created from raw data files using the Mascot Distiller software (version 2.1; MatrixScience, London, UK). The Mascot search algorithm (version 2.2) was used for searching against the International Protein Index database (release number IPI_rat—20091106.fasta). The peptide tolerance was typically set to 2 Da and the fragment ion tolerance to 0.8 Da. A maximum number of 2 missed cleavages by trypsin were allowed and carbamidomethylated cysteine and oxidized methionine were set as fixed and variable modifications, respectively. The Mascot score cut-off value for a positive protein hit was set to 31. Proteins present in the anti-Hu serum were omitted from the individual lists obtained by immunoprecipitation with the 4 anti-Tr positive sera. The remaining proteins of the 4 lists were compared by hand, leading to 12 proteins identified in all 4 anti-Tr immunoprecipitations. These were all further checked for absence in the list obtained by the 2 remaining negative controls (mGluR1, GluR1).
Culturing and Immunoblotting
HEK293T en HeLa cells were grown in DMEM:HAMF10 (1:1) medium containing 10% FBS and 1% penicillin/streptomycin at 37° C. and 5% CO2. Cells were transiently transfected with various DNA constructs using Polyethylenimin (Polyscience, Eppelheim, Germany) and grown for 24 hours after transfection. For glycosylation experiments, cells were treated with Tunicamycin (20 ug/ml, Merck Chemicals) for 24 hours prior to immunoblotting.
For immunoblotting cells were harvested in phosphate buffered saline (PBS) and lysed for 15 minutes at 4° C. in 1% Triton, 20 mM Tris pH7.5, 150 mM NaCl supplemented with complete protease inhibitors (Roche, Woerden, Netherlands). After a 15 seconds sonification step, samples were spun for 5 minutes at 13.000 rpm at 4° C. The supernatant was diluted in 2× Sample buffer (8% SDS/25% Glycerol/0.05M Tris pH 6.8/200 mM DTT/Bromophenol Blue/H2O) and separated on 8 or 10% SDS-PAGE gels, followed by blotting onto PVDF membranes (Biorad, Veenendaal, Netherlands). Blots were blocked with either 2% BSA/0.05% TWEEN®(polysorbate)/PBS (commercial antibodies) or with 0.05% TWEEN®/skimmed milk (patient sera) and incubated overnight at 4° C. with the appropriate antibody. Blots were washed with 0.05% TWEEN®/PBS or 0.05% TWEEN®/skimmed milk for 3 times 5 minutes each at room temperature and incubated with the appropriate secondary antibody. After washing 3 times in 0.05% TWEEN®/PBS and once in PBS, blots were developed with Enhanced Chemiluminescent Western Blotting Substrate (Pierce, Etten-Leur, Netherlands).
Immunocytochemistry
For immunocytochemistry, HeLa cells were grown on Lab-tek chamber slides (Nunc, Uden, Netherlands) and fixed with 4% PFA for 10 minutes, followed by 3 washes of 5 minutes each with PBS and 1 wash of 5 minutes with PBS+ (0.5% Protifar (Nutricia, Zoetermeer, Netherlands), 0.15% glycin, 0.05% Triton in PBS). Cells were stained with the primary antibody (2 hours at room temperature) in PBS+, washed 3 times in PBS and subsequently incubated with secondary antibodies in PBS+ for 1 hour at room temperature. After 3 subsequent washes in PBS slides were mounted in Vectashield (Vector Laboratories, Peterborough, UK) and studied on a DMRBE microscope (Leica, Rijswijk, Netherlands).
For surface staining of DNER, hippocampal neurons were incubated with serum of Tr9 diluted 1:50 and rabbit anti-HA diluted 1:100, in Neurobasal medium (NB) for 15 minutes at 37° C., followed by rinsing in NB at 37° C. After fixing the cells for 10 minutes in 4% PFA/4% sucrose cells were washed three times with PBS, followed by incubation with secondary antibodies in GDB (0.1% gelatin, 16.7 mM Phosphate buffer and 450 mM NaCl) for 1 hour at room temperature. After three subsequent washes in PBS the cover slips were mounted and studied as described above. Permeabilised staining of hippocampal neurons was performed by fixing the neurons in 4% PFA/4% sucrose, 3 washes with PBS and incubation of the primary antibodies in GDB+ 0.6% Triton, overnight at 4° C. After three washes with PBS, neurons were incubated with secondary antibodies for 1 hour at room temperature in GDB+ 0.6% Triton. Subsequent washing and mounting were as described above.
Immunohistochemistry
Snap frozen rat brain and cerebellar cryosections were stained as described before (Graus, F. et al., supra). Cryosections of 6-8 μm were air-dried and fixed in acetone (4° C.) for 10 minutes. Endogenous peroxidase was blocked with 0.3% H2O2 in PBS. Slides were treated with 10% goat serum for 30 minutes at room temperature (RT), incubated either 2 hours at RT or overnight at 4° C. with patient serum diluted in blocking serum (1:100 or 1:400), with PBS washes between the different steps. Slides were incubated with biotin conjugated goat anti-human IgG antibodies (1:2000, Vector, Peterborough, UK) in blocking serum for 45 minutes. After incubation with Vectastain Elite ABC complex (Vector Labs, Burlingame, Calif.) for 30 minutes, the reaction was developed with 0.05% diaminobenzidine and 0.01% H2O2 diluted in PBS with 0.5% Triton X-100 followed by staining with Diaminobenzidine tetrahydrochlorid dihydrate (DAB, Fluka, Zwijndrecht, Netherlands) slides were dehydrated with ethanol series (50-100%) and mounted with Pertex (Klinipath, Duiven, Netherlands). To test whether the different Tr-identified sera recognize a similar epitope, rat cerebellar sections were incubated with undiluted anti-Tr-positive serum for 3 hours at RT, after the initial blocking in goat serum. This was followed by overnight incubation at 4° C. with biotinylated purified IgG (biotinylated using EZ-Link® Sulfo-NHS-LC-Biotinylation Kit (Pierce Biotechnology, Rockford Ill., US), diluted 1:100 in PBS 1% BSA, from a serum which had been used in Tr-blocking experiments described before (Bernal, F. et al., 2003, Neurology 60:230-234). Staining was developed as described above.
Purification of DNER Reacting Antibodies from Tr Serum
BioGFP-DNER was expressed in HEK293T cells for 24 hours, washed with cold PBS and lysed for 15 min on ice in 20 mM HEPES pH7.5, 150 mM KCl, 1% Triton X-100, complete protease inhibitors (Roche, Woerden, Netherlands), followed by 2 times 10 seconds sonification steps. Extracts were spun at 13.000 rpm for 10 minutes after which the supernatant was incubated with preblocked (1% BSA, 0.05% TWEEN® in PBS for 30 minutes) Dynal M280 beads (Invitrogen, Breda, Netherlands) for 1 hour. Beads were washed 5 times with HEPES pH7.5, 600 mM KCl, 0.1% Triton X100 and subsequently incubated for 3 hours with patients sera diluted 1:15 in PBS containing 1% BSA and 0.05% TWEEN® at 4° C. The beads were washed 4 times with 20 mM HEPES pH7.5, 600 mM KCl, 0.1% Triton X100 and antibodies were eluted with 200 mM glycine pH2.8, after which the pH was neutralized using 1/10th volume of 1M Tris, pH8.0. Both depleted serum and the purified antibodies were washed with PBS using Vivaspin sample concentrators (GE Healthcare, Hoevelaken, Netherlands) and stored in PBS containing 1% BSA.7
DNER Knock Down in Hippocampal Neurons
Primary hippocampal cultures were prepared from embryonic day 18 (E18) rat brains9. Cells were plated on coverslips coated with poly-L-lysine (30 μg/ml) and laminin (2 μg/ml) at a density of 75,000/well. Hippocampal cultures were grown in Neurobasal medium (NB) supplemented with B27, 0.5 mM glutamine, 12.5 μM glutamate and penicillin/streptomycin as described in Jaworski et al. (supra). The following shRNA sequences targeting rat DNER were used in this study: shRNA#1 (AAACCCTTGGTCACACTGA) and shRNA#2 (GATTCTGTTGCTAAGTCCA). The complementary oligo's were annealed and inserted into pSUPER. Hippocampal neurons were transfected at 7 days in vitro (DIV) using Lipofectamine 2000 (Invitrogen). DNA (3.6 μg/well) was mixed with 3.3 μl Lipofectamine 2000 in 200 μl NB, incubated for 30 minutes and then added to the neurons in NB at 37° C. in 5% CO2 for 45 min. Next, neurons were washed with NB and transferred in the original medium at 37° C. in 5% CO2 for 4 days. Cells were co-transfected with β-galactosidase to identify the transfected cells.
Results
Identification of DNER as Tr-Antigen by Mass Spectrometry
To identify the antigen recognized by Tr-antibodies, we performed immunoprecipitation experiments followed by mass spectrometry on crude rat brain extracts using 4 different anti-Tr positive sera and 3 control sera containing antibodies against Hu, metabotropic glutamate receptor 1 (mGluR1) or glutamate receptor 1 (GluR1). Size fractionation of the purified complexes on SDS-PAGE electrophoresis and subsequent staining with Coomassie blue, showed no apparent protein band shared by all 4 anti-Tr positive sera (FIG. 1A). We nevertheless cut each lane in 15 slices, digested them with trypsin and performed mass spectrometry. The mass spectrometry data of each anti-Tr serum was first analyzed and compared to the results obtained with the Hu, mGluR1 and GluR1 control sera, leaving Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) as unique protein identified by the anti-Tr sera only, making DNER a good candidate as Tr-antigen.
DNER is the Tr-Antigen in Cell-Based Assay
We next performed a cell-based assay to find further evidence for DNER as the Tr-antigen. HeLa cells expressing full length HA-tagged DNER were incubated with anti-HA antibodies and either of two anti-Tr sera, an anti-Tr cerebrospinal fluid (CSF) and a control serum. The two anti-Tr sera and CSF strongly labeled cells expressing HA-DNER, but not untransfected cells (stained with Dapi only, FIG. 1B, top 3 panels). In contrast, the staining was absent in control sera known to detect Hu, mGLUR1 or GluR1 (FIG. 1B, lower panel and data not shown). We next tested all 12 anti-Tr sera as well as 246 control samples (Table 2), using the same cell-based screening assay. All anti-Tr sera positively stained HeLa cells expressing HA-DNER while all but one control samples were negative (FIG. 1B and data not shown). PAGE-7 Interestingly, the one control sample that contained anti-DNER antibodies was from a patient suffering from Hodgkin Lymphoma without any signs of ataxia. Testing this serum on rat brain sections resulted in the characteristic punctate staining typical for Tr (data not shown). As DNER is normally expressed in pyramidal hippocampal neurons, we performed co-staining in primary hippocampal neuron cultures. Under permeabilizing conditions, we detected punctuate somato-dendritic anti-DNER staining of endogenous DNER, which fully co-localizes with Tr-positive sera (FIG. 1D). These results further confirm that DNER is the Tr-antigen.
To determine whether the anti-Tr sera could detect the extracellular domain of DNER, we overexpressed HA-DNER in hippocampal neurons. Surface staining with both anti-HA and anti-Tr sera showed similar reactivity of both antibodies in the somato-dendritic compartment (not shown). This indicates that the DNER epitope is available to the anti-Tr antibodies on the surface of neurons.
DNER is the Tr-Antigen by Western Blot Analysis in Most but not all Anti-Tr Sera
Western blot analysis of cerebellar or purified Purkinje cell extracts with anti-Tr sera had so far not identified a common Tr antigen (Graus, F. et al., 1997, supra; Hammack, J. et al., 1992, Neurology 42:1938-1943). Since Western blot analysis is a more commonly used method for detecting autoimmune antibodies (Furneaux, H. M. et al., 1990, N. Engl. J. Med. 322:1844-1851) we tested whether this method could be used for anti-Tr diagnostic purposes. For this, we subjected total HEK293T cell extracts expressing HA-DNER and purified HA-DNER (by anti-HA IP, FIG. 1C) to Western blotting. Of the 12 anti-Tr positive sera tested only three could recognize total input directly (represented by Tr10 FIG. 1C). The other 9 sera were negative (represented by Tr9 in FIG. 1C). Loading the gel with purified HA-DNER, as seen by both anti-HA and anti-DNER antibodies (FIG. 1C) increased the number to 11 positive sera (see Tr9 FIG. 1C). However, serum Tr6, positive for DNER staining in the cell-based assay (FIG. 1B), remained negative (FIG. 1C). We therefore conclude that Western blotting is unreliable for diagnosing anti-Tr antibodies.
DNER Blocks Anti-Tr Antibody Reactivity on Brain Sections
Presence of anti-Tr antibodies is usually diagnosed by strict immunohistochemical criteria of rat frozen cerebellar sections: cytoplasmic labeling of Purkinje cells, combined with a characteristic punctuated somatodendritic staining in the molecular layer (Graus, F. et al., 1998, Acta Neuropathol. 96:1-7) (FIGS. 2A and 2A′). Staining rat brain slices with the commercial antibody against DNER resulted in the identical somatodendritic, punctuate staining of Purkinje cells (FIGS. 2B and 2B′). Both anti-Tr sera and anti-DNER antibodies stained isolated pyramidal neurons in the hippocampus (data not shown), as described before (e.g. Graus et al., 1997, supra). In all 7 cases analyzed with traditional antigen blocking experiments the tested anti-Tr sera abolished the characteristic-staining pattern of the standard anti-Tr serum (FIG. 2C-E).
In addition, we performed cerebellar immunohistochemistry with anti-Tr positive sera from which the anti-DNER antibodies were depleted. Sera incubated with purified biotinylated DNER (DNER-GFP-bio) coupled to streptavidin beads prior to the immunohistochemistry lost their Tr-characteristic staining of Purkinje cells (compare FIG. 2G to F). In contrast, eluted anti-DNER antibodies, showed staining of the Purkinje cells similar to the full serum (compare FIG. 2H to F).
Knock Down of DNER Removes Tr Staining in Hippocampal Neurons
To further demonstrate that DNER is the only protein detected by anti-Tr positive sera we performed DNER knock down experiments. Two independent shRNA-producing constructs directed against DNER were generated and transfected into hippocampal neurons. As endogenous DNER is expressed in pyramidal hippocampal neurons, we counted all transfected neurons as judged by presence of β-Galactosidase and checked for reactivity with anti-DNER and anti-Tr. In control transfections, 73% of the β-Galactosidase expressing neurons (118/162) were positive for both DNER and Tr (FIG. 3A, top panel). In contrast, transfection with two independent shRNA constructs targeting DNER removed both DNER staining, as observed by the commercial anti-DNER antibodies, and Tr staining (Tr9, FIG. 3A, lower panel) in all transfected neurones (133 and 134 neurons respectively for construct 1 and 2, FIG. 3B), further confirming the anti-Tr specificity for DNER.
Mapping of the Tr-Epitope in DNER
In order to identify the epitope recognized by Tr-positive sera (including the anti-Tr serum from the HL patient without ataxia), we generated different deletion mutants of DNER. We expressed all mutants in HeLa cells and scored whether the different anti-Tr sera reacted with DNER. All anti-Tr sera were found to recognize the extracellular domain of DNER, specifically the region between the second and third EGF-like domain (FIG. 4A, myc-DNER128-308). Interestingly, two different Tr-epitopes are identified within the 13 anti-Tr sera. 7 out of 13 sera recognized only the DNER region between amino acids 128-308 and were negative for the DNER fusion protein lacking this part (represented by Tr9 in FIG. 4A, HA-DNERΔ128-304). In addition, 6 sera recognized a second epitope within the DNER region of amino acid 302-675 and were positive for the DNER fusion protein lacking DNER128-308 (represented by Tr8 in FIG. 4A, myc-DNER302-675 and HA-DNERΔ128-304 respectively). An overview of the epitopes recognized by all 13 different anti-Tr sera using the cell-based assays is given in FIG. 4B. Recognition of the second epitope did not correlate with age, neurological symptoms, underlying type or stage of HL, or functional status of the patients (Table 1).
DNER Glycosylation is Required for Tr Antigenicity
DNER is highly glycosylated (Nishizumi, H. et al., 2002, Neuroreport. 13:909-915). To understand the role of glycosylation in the antigenicity of DNER, we incubated HEK293T cells, expressing HA-DNER without or with glycosylation inhibitor tunicamycin (20 μg/ml, for 24 hr) and analyzed anti-HA immunoprecipitated extracts using Western blot analysis. The removal of glycosylated chains in the majority of DNER protein was confirmed by the reduction in molecular mass of DNER to 90 kDa, close to the expected molecular mass (FIG. 4C). Although non-glycosylated DNER could still be recognized by the commercial anti-DNER antibody (data not shown), both anti-Tr positive sera tested no longer recognized the unglycosylated protein (FIG. 4C). This was confirmed by the lack of staining by anti-Tr sera of Hela cells expressing DNER constructs lacking the N-terminal signal peptide, which block DNER targeting into the endoplasmic reticulum and prevent glycosylation (data not shown). Glycosylation prediction software, NetNGlyc (www.cbs.dtu.dk/services/NetNGlyd) reveals the presence of 4 N-glycosylation sites in the common DNER epitope (amino acids 128-304). We mutated all 4 N-glycosylation sites into alanines (HA-DNERΔG1-4) and analyzed anti-Tr reactivity by immunoprecipitation followed by Western blotting (FIG. 4D). The commercial anti-DNER polyclonal antibodies recognized mutated DNER ΔG1-4, albeit with lower affinity. However, the 2 anti-Tr positive sera tested no longer recognized the mutated DNER (FIG. 4D, Tr9 and Tr10). The role of N-glycosylation for Tr antigenicity was further studied using the cell based assay in which we expressed myc-tagged DNER 128-308ΔG1-4. Two of the anti-Tr sera still recognize mutated DNER, but only very weakly (FIG. 4E, Tr5 and Tr8), whereas all other sera were negative PAGE-11 (FIGS. 4B and E). This strongly indicates the importance of N-glycosylation in recognition of the main DNER epitope by anti-Tr sera.
Applicants incorporate by reference the material contained in the accompanying computer readable Sequence Listing identified as P97562PC00_seqlist_ST25.txt, having a file creation date of Aug. 28, 2014, 10:31 a.m., and a file size of 10.5 kilobytes.







TABLE 1







Characteristics of 11 anti-Tr positive patients. All patients suffered severe cerebellar truncal


and limb ataxia with nystagmus and/or cerebellar dysarthria

























Time from















onset to













Anti-Tr

tumor


Ann










serum

detection

HL
Arbor HL
Tumor
Tumor

mRS



Pt No
Sex
Age
titer
MRI
(mo)
Tumor
subtype
Stage
treatment
response
Immunotherapy
worst
mRS end























1
M
73
6400
Normal
4.1
HL
NS
IIB
ABVD,
SD
PE
3
3











RT






2
M
71
400
Normal
2.9
HL
NS
IA
VAPEC-B
CR
—
4
4


3
M
50
1600
Normal
4.3
HL
NS
IA
EBVP
CR
PE,
4
4













steroids




4
F
65
>400
WML
—
No
—
—
—
—
—
5
6








tumor









6
F
55
>400
Old
7.8
HL
MC
IIIA
ABVD
CR
PE, IVIG
5
4






hemorrhage











7
M
36
1600
Normal
0.5
HL
NS
IIA
MOPP/ABV,
CR
—
2
2











RT






8
M
29
1600
Normal
1.0
HL
NS
IIIB
MOPP/ABV
CR
—
3
0


9
M
35
3200
Normal
4.8
HL
NS
IIB
EBVP,
CR
PE
4
4











RT






10
F
22
6400
Normal
4.6
HL
NS
IIA
ABVD,
CR
—
3
2











RT






11
M
54
>400
Cerebellar
10.7 
HL
NS
IIB
MOPP/ABV
CR
—
2
2






atrophy











12
F
12
>400
Normal
2.1
HL
NS
IIA
OEPA
CR
IVIG
4
3





MRI: WML, white matter lesions; Gd-enhancement, Gadolineum-enhancement; HL, Hodgkin Lymphoma;


HL subtype: NS, nodular sclerosing; MC, mixed cellularity; mRS, modified Rankin Score;


Tumor treatment: ABVD, adriamycin, bleomycin. vinblastine, dacarbazine; RT, radiotherapy; VAPEC-B, vincristine, adriamycin, prednisone, etoposide, cyclophosphamide, bleomycin; EBVP, epirubicin, bleomycin, vinblastine, prednisone; MOPP/ABV, mechlorethamine, Oncovin (vincristine), procarbazine, prednisone/adriamycin, bleomycin, vinblastine; OEPA, Oncovin (vincristine), etoposide, prednisone, adriamycin;


Tumor response: SD = stable disease, PD = progressive disease CR = complete response;


Immunotherapy: PE, plasma exchange; IVIG, intravenous immunoglobulins.













TABLE 2







Control samples








Sample
n











Blood bank controls
31


Hodgkin Lymphoma without ataxia
55


Hodgkin Lymphoma with paraneoplastic cerebellar ataxia
10


Non-Hodgkin Lymphoma without ataxia
14


Non-Hodgkin Lymphoma with paraneoplastic cerebellar ataxia
9


Ideopathic cerebellar ataxia
17


Other autoimmune disease:


SLE
10


Rheumatoid arthritis
20


Paraneoplastic cerebellar degeneration with known antibodies:


anti-Yo
20


anti-Ri
10


anti-Hu
20


anti-Ma
10


anti-Amphiphysin
10


anti-CV2
10


Total
246

## Claims

1. A method for determining the presence of anti-Tr antibodies in a subject comprising the steps of
(a) obtaining a sample from said subject, and
(b) testing for the presence of said antibodies in said sample by addition of glycosylated delta/notch-like epidermal growth factor related receptor (DNER) protein or an antigenic part thereof and checking whether said DNER protein or antigenic part thereof is bound by any anti-Tr antibodies in said sample.

2. The method according to claim 1, wherein the DNER protein or antigenic part thereof is labeled.

3. The method according to claim 1, wherein the sample is a blood, plasma, serum or cerebrospinal fluid (CSF) sample.

4. The method according to claim 1, wherein the method is an immunoassay.

5. The method according to claim 1, wherein the subject is a human subject.

6. The method of claim 1 wherein detection of said DNER protein or antigenic part thereof bound to said Anti-Tr antibodies is correlated with the presence of Hodgkin lymphoma.

7. The method of claim 1 further comprising typing subjects suffering from paraneoplastic cerebellar degeneration and/or ataxia based on detection of said DNER protein or antigenic part thereof bound to said Anti-Tr antibodies.

8. The method of claim 1 wherein a cell line transfected with a construct encoding the DNER protein or antigenic part thereof detects anti-Tr antibodies.

9. A kit for an immunoassay comprising
(a) a glycosylated delta/notch-like epidermal growth factor related receptor (DNER) protein or antigenic part thereof; and
(b) an anti-Tr antibody as positive control.

10. The kit according to claim 9, wherein the DNER protein or antigenic part thereof is labeled.

11. The method according to claim 4, wherein the immunoassay is a cell-based immunoassay.

12. The method according to claim 5, wherein the human subject is suffering from paraneoplastic cerebellar degeneration and/or ataxia.