# Patent

## Title
Fibroblast growth factor receptors as diagnostic markers of acquired sensory neuronopathies

## Abstract

The present invention relates to the diagnosis of acquired sensory neuronopathies (SNN), and to the treatment of these disorders.

## Background
FIELD OF THE INVENTION
The present invention relates to the diagnosis of acquired sensory neuronopathies (SNN), and to the treatment of these disorders.
BACKGROUND
Acquired sensory neuronopathies (hereinafter abbreviated as “SNN”) are a specific subgroup of peripheral nervous system diseases characterized by primary involvement of sensory neurons in the dorsal root ganglia (Kuntzer et al., Muscle Nerve, 2004; 30:255-268; Sghirlanzoni et al., Lancet Neurol., 2005; 4:349-361). SNNs encompass different paraneoplastic, viral, dysimmune, toxic, and idiopathic disorders.
Lesions in the dorsal root ganglia have been demonstrated pathologically in paraneoplastic SNN (Graus et al., Neurology, 1990; 40:219-222; Dalmau Jet al., Neurology 1991; 41:1757-1764; Wanschitz et al. Neurology, 1997; 49:1156-1159.), HIV infection (Scaravilli et al., Acta. Neuropathol. (Berl), 1992; 84:163-170; Esiri et al., J. Neurol. Sci., 1993; 114:178-187), Sjögren's syndrome (Mori et al., Brain, 2005; 128:2518-2534; Griffin et al., Ann. Neurol., 1990; 27:304-315), and unclassified connective diseases, but also in some idiopathic cases (Okajima et al., Neurology, 1983; 33:1061-1064; Sobue et al., Neurology, 1988; 38:463-467; Hainfellner et al., Ann. Neurol., 1996; 39:543-547; Kurokawa et al., J. Neurol. Neurosurg. Psychiatry, 1998; 65:278-279; Colli et al., Surg. Neurol., 2008; 69:266-273).
Recently, a set of clinical criteria that help to differentiate SNN from other sensory neuropathies has been published, but these criteria do not allow SNN to be distinguished according to their etiologies (Camdessanche et al., Brain, 2009; 132:1723-1733).
In particular, among SNN without an overt associated autoimmune context, it is at present not possible to distinguish on clinical grounds autoimmune SNN from non autoimmune idiopathic forms.
In addition, as these criteria were deliberately conceived to be stringent, they probably miss incomplete forms of SNN. As biopsy of dorsal root ganglia is not feasible as a routine investigation and because of the absence of methods that allow easy and non-traumatic exploration of dorsal root ganglia, there is a need for biological tools that may help to distinguish SNN from the far more numerous other form of sensory neuropathies.
Auto-antibodies reactive with sensory neuron antigens, mainly auto-antibodies called anti-Hu antibodies, have been identified in paraneoplastic SNN only (Camdessanche et al., Brain, 2009; 132:1723-1733; Graus et al., J. Neurol. Neurosurg. Psychiatry, 2004; 75:1135-1140; Camdessanche et al., Brain, 2002; 125:166-175; U.S. Pat. No. 6,193,948). A handful of studies using the serum of occasional patients with SNN and Sjögren's syndrome or idiopathic SNN tested on various substrates gave inconclusive results (Murata et al., Neuroreport., 2005; 16:677-681; Eystathioy et al., J. Mol. Med., 2003; 81:811-818; Dalakas M C, Ann. Neurol., 1986; 19:545-554; Nemni et al., Ann. Neurol., 1993; 34:848-854; van Dijk et al., J. Neuroimmunol., 1997; 74:165-172; Mutoh et al., Arch. Neurol., 2005; 62:1612-1615).
Differentiating SNN from other sensory neuropathies is important owing to the possibility of detecting disorders that may benefit from specific investigations and treatments.
Further, knowing whether an autoimmune process is involved in idiopathic cases or a subgroup of these is important, since it may lead to the development of immunomodulatory treatments and help to distinguish these cases from other sensory neuropathies.
The inventors have now shown that serum immunoreactivity toward fibroblast growth factor receptor (FGFR) family and related proteins, as well as toward proteins that belong to growth factor receptor-bound protein family, identify a subgroup of patients with SNN in which a dysimmune process is involved, suggesting that this subgroup of patients may be treated with immunosuppressants and/or immunomodulators.
DESCRIPTION OF THE INVENTION
In an attempt to find biomarkers which allow acquired sensory neuronopathies (SNN) to be distinguished from other neuropathies, the inventors unexpectedly found that a subgroup of patients who suffer from acquired sensory neuronopathy show an immunoreactivity toward a protein of the fibroblast growth factor receptor (FGFR) family, in particular toward FGFR3 (in particular toward the intracellular kinase domain of FGFR3, and/or TRK1 and/or TRK2 subunits of the intracellular kinase domain of FGFR3), FGFR1 and FGFR2, and to a lesser extent toward a protein that belongs to growth factor receptor-bound protein family, in particular growth factor receptor-bound protein 10 (GRB10). In this subgroup of patients suffering from SSN, most of the patients had no known associated autoimmune context.
Thus, the inventors have demonstrated for the first time that the presence in a sample from a patient of antibodies directed against proteins or fragments of proteins that belong to the fibroblast growth factor receptor (FGFR) family and/or against a protein or a fragment of a protein of the growth factor receptor-bound protein family can be used to discriminate between acquired sensory neuronopathies (SNN) and other neuropathies, in particular other sensory neuron disorders.
For instance, a patient diagnosed with a condition thought to be either proximal demyelinating polyneuropathy (abbreviated as “P-CIDP”) or SNN and another patient with a condition thought to be either a distal sensory neuropathy were firmly diagnosed as suffering from SNN after detection of anti-FGFR3 antibodies.
The inventors also noted that sera from some patients immunopositive toward FGFR3 showed a cross immunoreactivity toward FGFR1 and/or FGFR2.
The inventors further noted that the patients afflicted with SNN who show immunoreactivity toward a member of the FGFR family, in particular toward FGFR3, and/or toward a protein of the GRB family, have specific clinical characteristics which distinguish their neuronopathies from both anti-Hu associated SNN and FGFR3 sero-negative SNN. In particular, they are younger, more frequently women, the neuropathy has a progressive course, although a subacute and sometimes acute evolution is possible. Another striking clinical feature is the frequent trigeminal nerve involvement at onset and asymmetrical distribution of sensory manifestations at full development. Some patients may present with small fiber neuropathy or trigeminal nerve neuropathy.
Interestingly, FGFR3 antibodies were also detected in some patients suffering from autoimmune diseases, but without previously identified neurological disorders. When going back to their file, it was found that these patients with FGFR3 antibodies did in fact suffer from sensory neuron disorders, thereby strengthening the value of FGFR3 antibodies as biomarkers for the diagnosis of a subgroup of SNN.
SUMMARY OF THE INVENTION
Therefore, the present invention relates to a method of determining if a patient is afflicted with an acquired sensory neuronopathy, said method comprising:
a) detecting in a biological sample of the patient immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family; and optionally
b) deducing from the result of step a) whether the patient is afflicted with an acquired sensory neuronopathy, immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family is indicative of an acquired sensory neuronopathy.
The invention also relates to the use of antibody directed against a protein of the tyrosine kinase receptor family and/or of antibody directed against a protein of the growth factor receptor-bound protein family as a biomarker for diagnosing (or confirming) an acquired sensory neuronopathy in a patient.
The present invention also provides a method for selecting a patient afflicted with an acquired sensory neuronopathy suitable to be treated with at least one immunosuppressant and/or immunomodulator compound, comprising:
a) detecting in a biological sample of the patient immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family; and optionally
b) selecting the patient as suitable to be treated with at least one immunosuppressant and/or immunomodulator compounds when immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family is detected.
The above methods of the invention may be, for instance, in vitro or ex vivo methods.
The invention also concerns a method for treating a patient suffering from an acquired sensory neuronopathy who shows immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family, which method comprises administering to the patient immunosuppressant and/or immunomodulator compounds, or a pharmaceutical composition comprising said compounds.
The invention also provides immunosuppressant and/or immunomodulator compounds, or a pharmaceutical composition thereof, for use in the treatment of a patient who suffers from an acquired sensory neuronopathy and who shows immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family.
The invention further provides a kit for diagnosing an acquired sensory neuronopathy and a kit for selecting a patient afflicted with an acquired sensory neuronopathy suitable to be treated with at least one immunosuppressant and/or immunomodulator compounds, as described below.
It is to be understood that the methods, use and kits of the invention are not intended to identify or select patients suffering from any types of acquired sensory neuronopathy (SNN), but only to identify/select a specific subset of patients afflicted with an acquired sensory neuronopathy. It is not excluded that patients who are not identified by the methods, use and kits of the invention belong to another subset of patients afflicted with SNN.
DETAILED DESCRIPTION OF THE INVENTION
These and other objects, features and advantages of the invention will be disclosed in the following detailed description.
The present invention relates to a method of determining if a patient is afflicted with an acquired sensory neuronopathy, said method comprising:
a) detecting in a biological sample of the patient immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family; and optionally
b) deducing from the result of step a) whether the patient is afflicted with an acquired sensory neuronopathy, immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family is indicative of an acquired sensory neuronopathy.
The present invention also relates to the use of antibody directed against a protein of the tyrosine kinase receptor family and/or of antibody directed against a protein of the growth factor receptor-bound protein family as a biomarker for diagnosing (or confirming) an acquired sensory neuronopathy in a patient.
In a preferred embodiment, the patient to be tested is suffering, or is suspected to be suffering, from a neuropathy, preferably a sensory neuropathy.
A neuropathy is defined as a disorder of the peripheral nervous system, involving at least one of its motor, sensory or autonomic components. Each component includes the neuron cell body, its axons and the myelinating or non myelinating Schwann cells wrapping the axon. These components can be injured in the spinal cord anterior horns (motor neurons), in the dorasal root ganglia (sensory neurons) in the autonomic ganglia (autonomic neurons) and in the peripheral nerve including roots, plexus and nerves proper in which the axons circulate (see Dyck, Thomas, Lambert, Bunge. Peripheral Neuropathy. Vol. II. W.B. Saunders Co, Philadelphia, Pa. 1984).
A sensory neuropathy is defined as an exclusive or predominant involvement of sensory neurons with their axons and/or surrounding Schwann cells (see Dyck, Thomas, Lambert, Bunge. Peripheral Neuropathy. Vol. II. W.B. Saunders Co, Philadelphia, Pa. 1984). This disease includes sensory neuronopathies, distal or length dependent sensory neuropathies, small fiber neuropathies and sensory forms of chronic inflammatory demyelinating polyneuropathies, and focal sensory neuropathies such as trigeminal nerve neuropathies.
In another preferred embodiment, the patient to be tested is suspected to be suffering from an acquired sensory neuronopathies (SNN) and the method is performed to confirm that the patient is actually afflicted with this disease.
In a third embodiment, the patient to be tested is afflicted with lupus and/or Sjögren syndrome and is suffering from sensory symptoms or neuropathic pain and the method is performed to determine if the patient is actually afflicted with SNN.
The present invention also provides an in vitro method for selecting a patient afflicted with an acquired sensory neuronopathy suitable to be treated with at least one immunosuppressant and/or immunomodulator compound, comprising:
a) detecting in a biological sample of the patient immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family; and optionally
b) selecting the patient as suitable to be treated with at least one immunosuppressant and/or immunomodulator compounds when immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family is detected.
The method of determining if a patient is afflicted with an acquired sensory neuronopathy, the use of antibody directed against a protein of the tyrosine kinase receptor family and/or of antibody directed against a protein of the growth factor receptor-bound protein family as a biomarker for diagnosing (or confirming) an acquired sensory neuronopathy, and the method of selecting a patient afflicted with an acquired sensory neuronopathy suitable to be treated with at least one immunosuppressant and/or immunomodulator compound of the invention may be, for instance, in vitro or ex vivo methods.
The invention also concerns a method for treating a patient suffering from an acquired sensory neuronopathy who shows immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family, which method comprises administering to the patient immunosuppressant and/or immunomodulator compounds, or a pharmaceutical composition comprising said compounds.
The invention also provides immunosuppressant and/or immunomodulator compounds, or a pharmaceutical composition comprising said compounds, for use in the treatment of a patient suffering from an acquired sensory neuronopathy who shows immunoreactivity toward a protein of the tyrosine kinase receptor family and/or toward a protein of the growth factor receptor-bound protein family.
In some embodiments, the protein of the tyrosine kinase receptors family against which immunoreactivity is tested is a protein of the fibroblast growth factor receptor (abbreviated as “FGFR”) family or a protein of the tropomyosin-receptor-kinase (abbreviated as “Trk”) family.
In particular embodiments, the protein against which immunoreactivity is tested is a protein of the FGFR family chosen from the group consisting of receptors FGFR1 (e.g. NCBI accession number NP_075598.2 (SEQ ID NO: 1); NCBI accession number NP_075594.1 (SEQ ID NO: 2)), FGFR2 (e.g. NCBI accession number NP_000132.3 (SEQ ID NO: 3); NCBI accession number AAH39243 (SEQ ID NO: 4)), FGFR3 (e.g. NCBI accession number NP_000133; SEQ ID NO: 5) and FGFR4 (e.g. NCBI accession number NP_998812.1; SEQ ID NO: 6). In another particular embodiment, the protein against which immunoreactivity is tested is FGFR3.
The member of the FGFR family are structurally related proteins which exhibit an extracellular domain composed of three immunoglobin-like domains which form the ligand-binding domain, an acid box, a single transmembrane domain and an intracellular split tyrosine kinase domain. Multiple forms of FGFR-1 to -3 are generated by alternative splicing of the mRNAs. The extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation. This kinase domain interacts with different proteins responsible of the downstream effects of FGF fixation on their receptors. This includes proteins of the GRB family.
In another embodiment, the protein against which immunoreactivity is tested is a protein of the Trk family, preferably chosen from the group consisting of receptors TrkA (e.g. NCBI accession number NP_001007793.1, also known as “neurotrophic tyrosine kinase receptor type 1”; SEQ ID NO: 7), TrkB (e.g. NCBI accession number NP_001007098.1, also known as “neurotrophic tyrosine kinase receptor type 2”; SEQ ID NO: 8) and TrkC (e.g. NCBI accession number NP_001007157.1, also known as “neurotrophic tyrosine kinase receptor type 3”; SEQ ID NO: 9). Trk receptors are a family of tyrosine kinases that regulates synaptic strength and plasticity in the mammalian nervous system.
In another embodiment, the protein against which immunoreactivity is tested is a protein of the growth factor receptor-bound protein (abbreviated as “GRB”) family, preferably chosen from the group consisting of GRB1 to GRB14. More preferably, the protein of the growth factor receptor-bound protein family is GRB10 (for instance the GRB10 coded for by the polynucleotide sequence with NCBI accession number NM_001001550.1; SEQ ID NO: 10). GRB proteins are adaptor proteins involved in signal transduction triggered by activated tyrosine kinase receptors.
In a particularly advantageous embodiment, the protein(s) against which immunoreactivity is tested are at least FGFR3 and/or GRB10.
As used herein, reference to the “tyrosine kinase receptors”, the “FGFR receptors”, “Trk receptors”, and “GRB proteins” refer to all of the naturally-occurring variants, such as splice variants, allelic variants and isoforms, of said receptors or proteins.
In particular, the terms tyrosine kinase receptors, the FGFR, Trk receptor, or “GRB proteins” refer to:

    
    
        a) the polypeptide comprising or consisting of the amino acid sequence shown in NCBI accession number as recited above (for instances SEQ ID Nos: 1 to 10); and/or
        b) a polypeptide corresponding to the mature isoform of a polypeptide of (a) (i.e. obtained after cleavage of the signal peptide); and/or
        c) an allelic variant of a polypeptide of (a) or (b); and/or
        d) a splice variant of a polypeptide of (a), (b) or (c); and/or
        e) a constitutively active mutant of a polypeptide of (a), (b), (c) or (d).
        f) an isoform obtained by proteolytic processing of a polypeptide of (a), (b), (c), (d) or (e).
    
    


By “variant of a polypeptide” is meant a polypeptide that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a full-length polypeptide reference sequence. In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The <<needle>> program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk world wide web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS::needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.
As used herein, a “constitutively active mutant of a receptor (polypeptide) refers to a mutant of said receptor exhibiting a biological activity (i.e. triggering downstream signaling) in the absence of stimulation by its ligand, and/or exhibiting a biological activity which is higher than the biological activity of the corresponding wild-type receptor in the presence of its ligand.
As used throughout the present application, the expression “Immunoreactivity toward a target protein” (here a member of the receptor-tyrosine kinase family or a member of the growth factor receptor-bound protein family) is intended to mean that the sample from the patient to be tested comprises antibodies specifically directed against the target protein or a fragment of this target protein.
Therefore, immunoreactivity toward a target protein can be easily detected by demonstrating in the biological sample to be tested the presence of antibodies specifically directed against the target protein or a fragment of this target protein.
Fragments of the target proteins may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length protein. Preferably, said fragments are at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 250, 300, 350, 400, 450, 500 or more amino acids in length.
Such a test can be performed by one of ordinary skill in the art by using standard methods, for instance Enzyme-linked immunosorbent assay (“ELISA”), Western Blot/Dot Blot, Immunohistochemistry on transfected cells, Luminex (see for review Immunodiagnostics: A Practical Approach, R. Edwards Editor, Oxford University Press 2000; Manual of Molecular And Clinical Laboratory Immunology, J. D. Folds R. G. Hamilton, B. Detrick Editors ASM Press 2006; Immunology and Serology in Laboratory Medicine, M. L. Turgeon, Mosby Inc, 2008).
For instance, for determining the presence of anti-FGFR3 antibodies in a sample, the target protein can consist of, or comprise, the full-length FGFR3 receptor, the intracellular part of FGFR3, the intracellular kinase domain of FGFR3, the TRK1 and/or TRK2 subunits of the intracellular kinase domain of FGFR3, or a fragment thereof. Preferably the target protein consists of, or comprises, the intracellular kinase domain of FGFR3 (i.e. a fragment of a FGFR3 protein corresponding to fragment spanning the amino acid at position 397 or 399 to the amino acid at position 806, positions being given according to FGFR3 of NCBI accession number NP_000133), the TRK1 subunit of the intracellular kinase domain of FGFR3 (i.e. a fragment of a FGFR3 protein corresponding to fragment spanning the amino acid at position 403 to the amino acid at position 660, positions being given according to FGFR3 of NCBI accession number NP_000133), the TRK2 subunit of the intracellular kinase domain of FGFR3 (i.e. a fragment of a FGFR3 protein corresponding to fragment spanning the amino acid at position 661 to the amino acid at position 792, positions being given according to FGFR3 of NCBI accession number NP_000133) or a fragment thereof.
More preferably, the target protein consists of, or comprises, FGFR3 of NCBI accession number NP_000133 (SEQ ID NO: 5), TRK1 subunit of the intracellular kinase domain of FGFR3 of NCBI accession number NP_000133 (i.e. fragment 403-660 of FGFR3 of NCBI accession number NP_000133; SEQ ID NO: 12), or TRK2 subunit of the intracellular kinase domain of FGFR3 of NCBI accession number NP_000133 (i.e. fragment 661-792 of FGFR3 of NCBI accession number NP_000133; SEQ ID NO: 13).

Advantageously, the target protein consists of, or comprises, the intracellular kinase domain consisting of the amino acid sequence that spans amino acids 397 to 806 (SEQ ID NO: 11) or 399 to 806 of FGFR3 of NCBI accession number NP_000133 or a fragment thereof.

The terms “antibody” and “immunoglobulin” have the same meaning, and are used indifferently in the present invention.
The term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to specific epitope on the target protein.
In the context of the invention, the “biological sample” is preferably blood, serum, plasma or cerebrospinal fluid from the patient to be tested. More preferably, the biological sample is serum.
The term “immunosuppressant” (also called “immunodepressant”) refers to compounds that can suppress or prevent the immune response. Immunosuppressants are commonly used to prevent rejection of a transplanted organ and to treat autoimmune diseases, and are well known to a person of ordinary skill in the art. For instance, mention may be made of tacrolimus (CAS number 104987-11-3), cyclosporine (CAS number 59865-13-3), methotrexate (CAS number 59-05-2), glucocorticoids such as cortisol (CAS number 50-23-7), prednisone (CAS number 53-03-2), prednisolone (CAS number 50-24-8), methylprednisolone (CAS number 83-43-2), dexamethasone (CAS number 50-02-2), betamethasone (CAS number 378-44-9), cyclophosphamide (CAS number 50-18-0), azathioprine (CAS number 446-86-6), mycophenolate mofetil (CAS number 24280-93-1), plasma exchanges, intravenous immunoglobulins, compounds that target B cells (e.g. anti-CD20 antibodies) or T-cells activation, compounds (for instance fingolimod, CAS number 162359-55-9) that block efference from lymphoid organs (i.e. migration of mature leukocytes/lymphocytes from lymphoid organs) (see for instance Umapathi T, Hughes R A, Nobile-Orazio E, Léger J M, Immunosuppressant and immunomodulatory treatments for multifocal motor neuropathy, Cochrane Database Syst Rev. 2012 Apr. 18; 4:CD003217; Dalakas M C; Medscape, Advances in the diagnosis, pathogenesis and treatment of CIDP, Nat Rev Neurol. 2011 Aug. 16; 7(9):507-17; Kieseier B C, Lehmann H C, Meyer Z u Hörste G, Autoimmune diseases of the peripheral nervous system, Autoimmun Rev. 2012 January; 11(3):191-5; Walgaard C, Jacobs B C, van Doorn P A, Emerging drugs for Guillain-Barré syndrome, Expert Opin Emerg Drugs. 2011 March; 16(1):105-20; Hutton E J, Lunn M P, Treatment in inflammatory neuropathies. Expert Rev Clin Immunol. 2010 March; 6(2):231-45; Tracy J A, Dyck P J, Investigations and treatment of chronic inflammatory demyelinating polyradiculoneuropathy and other inflammatory demyelinating polyneuropathies, Curr Opin Neurol. 2010 June; 23(3):242-8).
As used herein, the term “patient” denotes a human being.
In the context of the invention, the term “treating” is used herein to characterize a therapeutic method or process that is aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) alleviating or bringing about ameliorations of the symptoms of the disease state or condition to which such term applies; and/or (3) reversing or curing the disease state or condition to which such term applies.
The immunosuppressants and immunomodulators used in the above recited method or use for treating patients afflicted with SNN are provided in a pharmaceutically acceptable carrier, exipient or diluent which is not prejudicial to the patient to be treated.
Pharmaceutically acceptable carriers and exipient that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
As appreciated by skilled artisans, compositions are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral route, including for instance intramuscular, subcutaneous, intravenous, intraperitoneal or local intratumoral injections. The oral route can also be used, provided that the composition is in a form suitable for oral administration, able to protect the active principle from the gastric and intestinal enzymes.
Further, the amount of immunosuppressants and/or immunomodulators used in the above recited method or use for treating patients afflicted with SNN is a therapeutically effective amount. A therapeutically effective amount of immunosuppressants and/or immunomodulators is that amount sufficient to suppress (by at least about 10%, preferably by at least about 30%, preferably by at least about 50%, preferably by at least about 70, 75 or 80%, still preferably by 85, 90, 95, or 100%) the immune response, preferably the immune response specifically directed toward tyrosine kinase receptors, the FGFR, Trk receptor, and/or “GRB proteins” against which the patient shows immunoreactivity, or to treat a desired disease without causing overly negative effects in the subject to which the immunosuppressants and/or immunomodulators are administered.
The exact amount of immunosuppressants and/or immunomodulators to be used and the composition to be administered will vary according to the age and the weight of the patient being treated, the type of disease, the mode of administration, the frequency of administration as well as the other ingredients in the composition which comprises the immunosuppressants and/or immunomodulators. Such concentrations can be routinely determined by those of skilled in the art. The amount of the immmunoglobulin actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual immunosuppressants and/or immunomodulators administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, etc.
Generally, the immunosuppressants and/or immunomodulators used in the above recited method or use for treating patients afflicted with SNN may be administered in the typical range. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. For instance, typical dose of prednisone can be 1 mg/kg/day, that of tacrolimus 0.10-0.20 mg/kg/day, that of cyclosporine 2-6 mg/kg/day, that of methotrexate 7.5-15 mg/week, that of cyclophosphamide 500 mg/m2 every month, that of azathioprine 150 mg/day, that of micophenolate mophetil 2 g/day, that of intravenous immunoglobulins 0.4 g/kg/day for 4 days, that of or rituximab 375 mg/m2 every week for four weeks or 2 infusions of 1000 mg at 2 weeks interval.
The invention further provides kits that are useful in the above methods for diagnosing an acquired sensory neuronopathy or for selecting a patient afflicted with an acquired sensory neuronopathy suitable to be treated with at least one immunosuppressant and/or immunomodulator compounds.
Such kits comprise means for detecting antibodies directed toward at least one protein of the tyrosine kinase receptor family and/or toward at least one protein of the growth factor receptor-bound protein family.
Preferably, the kit comprises at least means for detecting antibodies directed toward FGFR3, the intracellular part of FGFR3 and/or the intracellular kinase domain of FGFR3, and/or GRB10 (preferably a GRB10 protein coded for by the polynucleotide sequence SEQ ID NO: 10). Still preferably, in one embodiment the kit consists of means for detecting antibodies directed toward FGFR3, the intracellular part of FGFR3 and/or the intracellular kinase domain of FGFR3 and/or the TRK1 and/or TRK2 subunits of the intracellular kinase domain of FGFR3, and means for detecting antibodies directed toward GRB10. Advantageously, the intracellular kinase domain of FGFR3 consists of the amino acid sequence that spans amino acids 397 to 806 (SEQ ID NO: 11) or 399 to 806 of FGFR3 of NCBI accession number NP_000133), the TRK1 subunit of the intracellular kinase domain of FGFR3 of NCBI accession number NP_000133 (i.e. fragment 403-660 of FGFR3 of NCBI accession number NP_000133; SEQ ID NO: 12), the TRK2 subunit of the intracellular kinase domain of FGFR3 of NCBI accession number NP_000133 (i.e. fragment 661-792 of FGFR3 of NCBI accession number NP_000133; SEQ ID NO: 13).
Such means can be the target protein(s), i.e. the protein(s) of the tyrosine kinase receptor family and/or of the growth factor receptor-bound protein family against which immunoreactivity is tested, or fragments thereof as described above.
For instance, when immunoreactivity toward FGFR3 is tested, the target(s) protein(s) is (are) preferably chosen from the group consisting of i) the full-length FGFR3 receptor, ii) a protein which consists of, or comprises, the intracellular kinase domain of FGFR3, iii) a protein which consists of, or comprises, the TRK1 subunit of the intracellular kinase domain of FGFR3, and iv) a protein which consists of, or comprises, the TRK2 subunit of the intracellular kinase domain of FGFR3, more preferably the target(s) protein(s) is (are) chosen from the group consisting of i) FGFR3 of NCBI accession number NP_000133 (SEQ ID NO: 5), ii) a protein which consists of, or comprises, the intracellular kinase domain consisting of the amino acid sequence that spans amino acids 397 to 806 (SEQ ID NO: 11) or 399 to 806 of FGFR3 of NCBI accession number NP_000133, iii) a protein which consists of, or comprises, the TRK1 subunit of the intracellular kinase domain of FGFR3 of NCBI accession number NP_000133 (i.e. fragment 403-660 of FGFR3 of NCBI accession number NP_000133; SEQ ID NO: 12), and iv) a protein which consists of, or comprises, the TRK2 subunit of the intracellular kinase domain of FGFR3 of NCBI accession number NP_000133 (i.e. fragment 661-792 of FGFR3 of NCBI accession number NP_000133; SEQ ID NO: 13). In a preferred embodiment, when immunoreactivity toward FGFR3 is tested, the target(s) protein(s) comprise(s) at least a protein which consists of, or comprises, the intracellular kinase domain consisting of the amino acid sequence that spans amino acids 397 to 806 (SEQ ID NO: 11) or 399 to 806 of FGFR3 of NCBI accession number NP_000133.
For instance, when immunoreactivity toward GRB10 is tested, the target protein consists of all or part of GRB10, preferably all or part of a GRB10 protein coded for by the polynucleotide sequence SEQ ID NO: 10.
For instance, when immunoreactivity toward FGFR1 is tested, the target protein consists of all or part of FGFR1, preferably all or part of a FGFR1 protein of SEQ ID NO: 1 or of SEQ ID NO: 2.
When immunoreactivity toward FGFR2 is tested, the target protein, for instance, consists of all or part of FGFR2, preferably all or part of a FGFR2 protein of SEQ ID NO: 3 or of SEQ ID NO: 4.
Means for detecting antibodies directed toward at least one protein of the tyrosine kinase receptor family and/or toward at least one protein of the growth factor receptor-bound protein family may also include an antibody specifically binding to human antibodies (used as a “secondary antibody” which binds to antibodies from the sample to be tested specifically binding to the target protein). Such antibodies can be labeled with detectable compound such as fluorophores or radioactive compounds.
In a preferred embodiment, the kit according to the invention may further comprises a control sample comprising a known amount of antibodies and/or instructions for the use of said kit in diagnosing an acquired sensory neuronopathy or in selecting a patient afflicted with an acquired sensory neuronopathy suitable to be treated with at least one immunosuppressant and/or immunomodulator compounds.
The means may be present, e.g., in vials or microtiter plates, or be attached to a solid support. For instance the target protein can be attached to a membrane or to an array.
All references cited herein, including journal articles or abstracts, published patent applications, issued patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references.
The invention will be further evaluated in view of the following examples and figures.



DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the level of reactivity of 16 non paraneoplastic SNN and 30 controls on FGFR3. The dotted line indicates the cut-off level. Protoarray assay
FIG. 2 illustrates the level of reactivity of 16 non paraneoplastic SNN and 30 controls on GRB10. The dotted line indicates the cut-off level. Protoarray assay.
FIG. 3 is a histogram which illustrates the results of ELISA assays carried out on sera from patients suffering from autoimmune diseases or neuropathies, with the human FGFR3 399-806 aa peptide corresponding to the protein intracellular domain.
Comparison of the mean OD (optical density) minus blank value in the groups (ANOVA test) is presented: D=blood donors; lupus/SGS=autoimmune diseases; other neurop=other neuropathies; SNN=sensory neuronopathies. The patient and control groups are on the x-axis and the OD values minus blank values are on the y-axis.
FIG. 4 is a histogram which illustrates the results of ELISA assays carried out on sera from patients suffering from autoimmune diseases or neuropathies, with the human FGFR3 399-806 aa peptide corresponding to the protein intracellular domain.
Comparison of the normalized OD in four groups (ANOVA test) is presented: D=blood donors; lupus/SGS=autoimmune diseases; other neurop=other neuropathies; SNN=sensory neuronopathies. The patient and control groups are on the x-axis and the normalised OD values are on the y-axis.
FIG. 5 illustrates the results of ELISA assays with the human FGFR3 399-806 aa peptide corresponding to the protein intracellular domain.
Part A: normalised OD.
Part B: index.
D=blood donors; lupus/SGS=autoimmune diseases; other neurop=other neuropathies; SNN=sensory neuronopathies.
The dotted line indicates the cut-off level.



EXAMPLES
Example 1
Materials and Methods
1.1. Serum Samples
Serum samples from 261 individuals were collected. Sera were snap frozen at −80° C. and stored until utilisation. Individuals were classified into four groups. Group I corresponded to 59 healthy blood donors. Group II included 56 patients with systemic autoimmune diseases and lupus or Sjögren syndrome antibodies, group III included 104 patients with a diagnosis of sensory neuropathy, and group IV included 42 patients with sensorimotor peripheral neuropathies. Among group III, the neuropathy was a probable or possible SNN according to published criteria in 85 patients (Camdessanche J. P. et al., Brain 2009; 132:1723-1733). Among the 19 who did not fill these criteria, 7 had small fiber SNN and 2 a sensory neuropathy that could not be distinguished between proximal demylinating polyneuropathy (P-CIDP) and SNN. In the other, the criteria were not fulfilled mainly because ENMG sensory nerve abnormalities were mild. A known dysimmune context was associated with the neuropathy in 72/104 patients including, Sjögren syndrome, lupus, lupus anticoagulant, unclassified connective disorders, monoclonal gammopathy, including CANOMAD syndrome, and HIV infection. Eight had paraneoplastic SNN with Hu antibodies. Patients in group IV had Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, neuropathy with monoclonal gammopathy, mononeuritis multiplex with vasculitis, diabetic neuropathy, hereditary neuropathy or idiopathic length dependent axonal neuropathy. As a whole the neuropathy was dysimmune in 11/42.
1.2. Protein Arrays
Sera were probed in the human ProtoArray v.4.2 (Invitrogen, Carlsbad, N. Mex., USA). These microarrays contained 9000 human GST-tagged proteins, expressed in Sf9 insect cells and spotted in duplicate. Protoarrays were used according to the recommendations of the manufacturer. Briefly, the slides were equilibrated at 4° C. for 15 min and then incubated with blocking buffer (50 mM Hepes, pH7.5, 200 mM Nacl, 0.08% Triton X-100, 25% Glycerol, 20 Mm Reduced glutathione, 1 mM DTT, 1X Roti-Block) for 1 h at 4° C. with gentle shaking. Then slides were incubated with human sera diluted 1:500 in washing buffer (1X PBS, 0.1% Tween 20, 1X Roti-Block) for 90 min at 4° C. with gentle shaking. Slides were washed five times with the washing buffer and incubated with 1 μg/ml of secondary antibodies (Alexa Fluor 647 goat anti-human IgG antibody; Invitrogen) to detect the Human bound antibodies. The arrays were washed and dried by centrifugation at 200 g for 1 min. An array used as a control was incubated with the secondary antibody for background determination. A protein gradient of purified human IgG printed on each subarray was used as a positive control. Finally, the slides were scanned using a Genepix 4000B scanner (Axon, Union City, Calif., USA) with the laser set at 635 mm, the laser power at 100%, and the photomultiplier gain at 800. GenePix Pro 3.0 image analysis software (Axon, Union City, Calif., USA) was used for the quantification. The ProtoArray Prospector v5.0 software was used to identify immunoreactivities. This software uses the Chebyshev inequality P value, which is derived by testing the null hypothesis. Two statistical tools were used to detect positive spots, CI p-value and z-score. They correspond respectively to the probability that a spot is similar to a negative control, and to the spot signal minus average (for all spots) divided by the standard deviation for all spots. The NNS serum samples were compared versus control serum samples to obtain significant protein hits. The software also calculates the z score for each printed spot's fluorescent intensity. The z score indicates the deviation of each protein's antibody reading from its distribution mean (SD).
1.3. Dot Blot
A dot blot was used to assess the ability of target proteins to screen for SNN status in serum. Two to 10 μl of the purified recombinant peptide consisting of the 399-806 aa of the protein corresponding to the intracellular kinase domain of human FGFR3 (Invitrogen) used for the ProtoArray was spotted in nitrocellulose membrane. The membrane dried for 5 min and was blocked with 5% BSA in TBS-T for 1 h. Then serum samples (dilution 1/50 in blocking buffer) were incubated overnight at 4° C. After washing three times, biotinylated goat anti anti-human IgG antibody (Dako, Glostrup, Denmark) diluted 1:100 in blocking buffer, followed by streptavidin-peroxidise was used. Immunoreactive spots were visualized using diaminobenzidine (DAB) detection reagents. As control, a rabbit polyclonal anti-FGFR3 antibody directed against the 742-806 aminoacide sequence of human FGFR3 (GeneTex, San Antonio, Tex., USA) was used.
1.4. ELISA
The ELISA method was used to assess the ability of target protein identified by protoarray analysis to screen for sensitive ganglionopathy status in serum. Briefly, microtiter plates (Maxisorp, Nunc) were coated overnight at 4° C. with 1 μg/ml of the purified recombinant intracellular domain of human FGFR3 (Invitrogen), the full length recombinant FGFR1 (NCBI Reference Sequence: NP_075594.1; SEQ ID NO: 2) or FGFR2 proteins (AAH39243; SEQ ID NO: 4 (Novus biologicals) in carbonate-bicarbonate 0.05 M pH 9.6 solution. After washing one time with washing buffer (PBS containing 0.1% Tween 20), plates were blocked with blocking buffer, washing buffer containing 3% SVF and 0.1% gelatine for 1 h at room temperature. Then, serum samples (dilution 1/50 in blocking buffer) were incubated for 1 h at room temperature. After washing four times with washing buffer, anti-human IgG peroxidase-labelled (dilution 1/3000 in blocking buffer) was added for 1 h à room temperature. Then the signal was developed with o-Phenylenediamine substrate for 15 min to 30 min (sigma) and read at 450 nm. Each serum was tested in duplicate and the mean optic density (OD) of the readings was taken into account for the analysis. Controls included blank wells containing the products of the reaction minus human sera and the secondary antibody. The rabbit polyclonal anti-FGFR3 antibody diluted at 1/1000 and the appropriate secondary antibody were used as control and to normalise readings among plates. The specific reactivity of patients or controls' samples was obtained by subtracting the readings of plates of a pool of sera from a panel of healthy blood donors from the readings of plates of tested sera (hereafter designated as the normalized OD). In addition to limit variation among tests an index was built as follows: (OD of the tested serum/OD of the blank wells)/(OD of the pool of control sera/OD of the blank wells). To be considered as positive, the serum must be positive both for the normalized OD and the index.
1.5. Expression of FGFR by Sensory Neurons.
Purified cultures of dorsal root ganglia (DRG) neurons were established from embryonic day 18 (E18) rats as previously described (Seilheimer B. et al., J. Cell. Biol. 1988; 107:341-351). Briefly, DRG were collected in L-15 medium (Gibco Invitrogen) containing antibiotics, centrifuged, and incubated for 15 min in 0.25% EDTA-trypsin (Gibco Invitrogen), then centrifuged and resuspended in MCM. The pellets were mechanically dissociated by passage through a 21 gauge needle and the neurons plated on poly-L-lysine-coated coverslips in MCM containing 50 ng/mL of recombinant human b-nerve growth factor (NGF, Peprothech, Rocky Hill, N.J., USA) and 5 mg/mL of glucose. After two days of cultures cells were fixed for 3 minutes in 5% paraformaldehyde and tested by immunohistochemistry with either anti-FGFR3, FGFR1, FGFR2 (GenTex), CRMP5 (home made) antibodies diluted at 1/500 and revealed either with FITC-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) at a 1:1000 dilution or rhodamine-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich) at a 1:2000 dilution. Double-immunolabelling was performed by incubating the sample with the primary antibodies under the conditions described above, followed by the appropriate FITC- and rhodamine-conjugated antibodies.
1.6. Immunocytochemistry on HEK293 Cells.
HEK293 cells were transfected with plasmids containing the full length (pEGFPN3-FGFR3 full lengh), the intracellular domain (i.e. fragment which spans amino acids 397 to 806 of FGFR3) (pEGFPN3-FGFR3 cytoplasmic domain), the TRK1 (pEGFPN3-FGFR3-TK1 domain) or the TRK2 (pEGFPN3-FGFR3-TK2 domain) subunits of the intracellular domain of human FGFR3 tagged with EGFP or plasmid without insert (control).
The plasmids pEGFPN3-FGFR3 full lengh, pEGFPN3-FGFR3 cytoplasmic domain, pEGFPN3-FGFR3-TK1 domain and pEGFPN3-FGFR3-TK2 domain were amplified from pcDNA3-hFGFR3 (a generous gift from Dr. Vigdis Sorensen, University of Oslo, Norway) by the following primers:

    
    
        5′ATGGGCGCCCCTGCCTGC3′ (SEQ ID NO: 14) and 5′CGTCCGCGAGCCCCCAC3′ (SEQ ID NO: 15) for pEGFPN3-FGFR3 full lengh.
        5′ATGAAGAAAGGCCTGGGCTCC3′ (SEQ ID NO: 16) and 5′CGTCCGCGAGCCCCCAC3′ (SEQ ID NO: 17) for pEGFPN3-FGFR3 cytoplasmic domain
        5′ATGAAGAAAGGCCTGGGCTCC3′ (SEQ ID NO: 18) and 5′CCACTTCACGGGCAGCC 3′ (SEQ ID NO: 19) for pEGFPN3-FGFR3-TK1 domain
        5′ATGGCGCCTGAGGCCTTG3′ (SEQ ID NO: 20) and 5′CGTCCGCGAGCCCCCAC3′ (SEQ ID NO: 21) for pEGFPN3-FGFR3-TK2 domain
    
    


The PCR product was cut with Hind III and BamH1 and ligated into EGFP-N3 vector (Clontech laboratories).
HEK293 cells were cultured in Dulbecco's modified minimal essential medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and antibiotics (25 U/ml penicillin and 25 μg/ml streptomycin and amphotericin) at 37° C. with 5% CO2. The day before transfection, the HEK293 cells were seeded into 24 well culture plates (9.104 per well).
HEK293 cells were transfected using lipofectamine LTX (Invitrogen). A 100 μl mix containing 0.5 μg plasmidic DNA and 1.5 μl lipofectamine in OPTI-MEM medium (Invitrogen) was added to each well for 48 h at 37° C.
Cells were then fixed in 4% paraformaldehyde for 5 min, washed with PBS. The cells were then blocked and permeabilized with 0.2% gelatin buffer containing 0.1% triton X-100 at room temperature for 1 h before being incubated overnight at 4° C. with patient's sera (diluted 1/20). After incubation, cells were rinsed with PBS and incubated with TRITC-goat anti-human IgG (Interchim) diluted 1/2000 for 3 h at 4° C.
Immunostaining was observed with Zeiss fluorescence microscope.
Example 2
Results
2.1. Protein Arrays
46 serum samples from 16 non paraneoplastic SNN (4 with associated dysimmune disorders) and 30 controls (15 healthy blood donors, 8 anti-Hu associated SNN and 7 non SNN neuropathies) were probed in the human ProtoArray v.4.2 (Invitrogen). Using the ProtoArray Prospector v5.0 software, CI p-value and z-score with the respective standard cut-off of 0.001 and 4, 442 immunoreactivities were identified as significantly different in the SNN group versus the control group. Using the following stringent criteria: reactivity restricted to the SNN group, level of reactivity >1010 in the SNN group and <900 in the control group and Z score >5, only two immunoreactivities distinguished significantly the SNN group from the control one: namely anti-FGFR3 (NCBI Reference Sequence: NP_000133.1) and GRB10 (NCBI Reference Sequence: NM_001001550.1) reactivity present in 7/16 patients respectively (cf. FIGS. 1 and 2). Six sera reacted with both FGFR3 and GRB10 and one with each of them respectively so that 8 sera reacted with at least one of them. The level of reactivity was wholly higher with FGFR3 than with GRB10.
2.2. ELISA with the FGFR3 Protein.
152 out of the 271 sera (patients or controls) were tested from 2 to 10 times, median 3, and used for adjusting the method.
By an ANOVA test, the OD minus the blank value of sample sera was significantly higher in the sensory neuropathy and autoimmune disease groups comparatively to blood donors and patients with other peripheral neuropathy (FIG. 3). By the same test, patients with sensory neuropathy had higher normalized OD comparatively to the other groups (FIG. 4).
ROC curves were used to determine the cut-off values of the normalised OD and the index discriminating the SNN group from blood donors and other peripheral neuropathies with 100% specificity. A value of 0.13 for the normalized OD and 1.55 for the index were determined as the respective cut-off values. To be considered as positive for anti-FGFR3 antibodies both the normalized OD and the index values of a given serum had to be superior to the cut-off levels.
This allowed the identification of 14/104 patients with sensory neuropathy, 0/59 blood donors, 0/42 patients with other neuropathies and 5/56 patients with autoimmune diseases with FGFR3 antibodies (FIG. 5). The neuropathy in group III, was associated to an autoimmune context in 3/14 patients and diagnosed as possible or probable SNN in 13/14 cases the last patients having a form difficult to differentiate between SNN and P-CIDP. When going back to the clinical file of the 5 patients with autoimmune disease (group II), two of them proved to have trigeminal sensory neuronopathy, one sensory neuropathy in the four limbs, one chronic thoracic neuropathic pain and one no known neurological syndrome.
2.3. Dot Blot with FGFR3
To confirm the Elisa result by another method, the commercial FGFR3 antibody and two sera with patients with anti-FGFR3 antibody reacted by dot blot with the FGFR3 protein while sera from a blood donor did not (data not shown).
2.4. Elisa with FGFR1 and FGFR2 Proteins
As the FGFRs proteins share a high degree of homology, to test the possibility of serum cross immunoreactivity between FGFR3 and the other FGFRs, a sample of SNN, autoimmune disease and control sera were tested by Elisa with the FGFR1 and 2 recombinant proteins. FGFR4 was not tested as none of the SNN and control sera analysed by the protoarray method reacted with it. Four out of 13 sera positive for FGFR3 were found positive with FGFR1 while none of 14 sera negative for FGFR3 were positive for FGFR1. Concerning FRGF2, 2/7 sera positive for FGFR3 were positive for FGFR2 while none of 5 sera negative for FGFR3 were positive for FGFR2. Two sera reacted with the three proteins. These results show that although cross immunoreactivity may occur with FGFR 1 and 2, FGFR3 is more frequently recognized by autoantibodies in patients with sensory neuronopathy.
2.5. Expression of FGFR Proteins by Sensory Neurons In vitro.
As the patients with anti FGFR3 immunoreactivity were thought to have primary sensory neuron involvement that might be mediated to an auto-immunoreactivity directed toward FGFR3, the inventors checked that FGFR3 was expressed by dorsal root ganglia sensory neurons. By immunohistochemistry the FGFR3, 2 and 1 antibodies immunolabelled the cytoplasm of sensory neurons double labelled with the CRMP5 antibody while the FGFR5 antibody immunolabelled neuron nuclei (data not shown)).
2.6. Clinical Characteristics of the Neuropathy in Patients with FGFR3 Immunoreactivity.
As 17 of the 19 patients with FGFR3 antibody had sensory peripheral neuropathy (89%) multivariate logistic regression was used to compare the clinical and electrophysiological characteristics of their neuropathy with that of 36 patients without anti-FGFR3 antibody and non paraneoplastic sensory neuropathy and 31 patients with anti-Hu antibody and paraneoplastic SNN (see Table 1 below). The clinical and electrophysiological items analyzed in this study have been published elsewhere (Camdessanche J. P. et al., Brain 2009; 132:1723-1733).







TABLE 1







comparative clinical manifestation in patients with FGFR3


positive, FGFR3 negative and anti-Hu positive sensory neuropathy.











FGFR3 positive
FGFR3 negative
Hu positive














Number
19
38
31


Age (M + SD)
49.1 ± 15.6
60.5 ± 13.9
61.6 ± 11.3


Sex (female)
70%
50%
19%


Dysimmune contex
42%
26%
—







ONSET










Acute
17%
12%
36%


Subacute
22%
22%
48%


Progressive
61%
66%
16%


Face
28%
3%
0%


Lower limbs
56%
71%
58%


Upper limbs
39%
45%
80%







FULL DEVEVELOPMENT










Lower limbs
78%
95%
97%


Upper limbs
78%
71%
94%


Face
28%
11%
13%


Trunk
17%
11%
10%


Dysautonomia
22%
27%
23%


Pain
55%
46%
58%


Ataxia
50%
62%
74%


Assymetry
56%
17%
48%







CSF










Normal
50%
44%
0%









Comparatively to patients without FGFR3 antibody and no paraneoplastic sensory neuropathy, patients with FGFR3 antibody tended to be younger (OR 0.97:0.93-1.02 95% CI; p=0.06), had more frequent trigeminal nerve involvement at onset of the neuropathy (OR 27.6:2.3-331.1 95% CI; p=0.009) and asymmetrical distribution of sensory loss at full development of the disorder (OR 8.7:1.9-41.2 95% CI; p=0.006). Comparatively to anti-Hu paraneoplastic SNN, patients with FGFR3 antibody were younger (OR 0.94:0.90-0.99 95% CI; p=0.02) and less frequently male (OR 0, 13:0.03-0.50 95% CI; p=0.003). Their neuropathy tended to have a more frequent slow progressive course (OR 7.2:0.9-62.9 95% CI; p=0.06) and less frequent ataxia (OR 0.04:0.001-1.1 95% CI; p=0.06). Interestingly none of the electrophysiological criteria used in this study (number of abolished sensory action potentials or motor nerve conduction abnormalities) discriminated patients with FGFR3 antibody from those without whether their neuropathy was paraneoplastic or not. Nine patients with FGFR3 antibody underwent a spinal tap. A mild elevation of protein concentration was observed in 3, oligoclonal bands in 3 and cell reaction in 3. As a whole the CSF was abnormal in 5/9. A nerve biopsy was available in 4 patients (1 with Sjögren syndrome and 3 without known autoimmune context) and showed fiber loss without regenerating cluster consistent with a diagnosis of SNN in all of them. In one patient without autoimmune context, nerve vasculitis consisting of CD3+T lymphocytes was present in the epineurium.
2.7. Reactivity of Sera with HEK FGFR3 Transfected Cells.
Serum samples from four patients with sensory neuronopathy and anti-FGFR3 antibody detected by ELISA and four healthy blood donors were tested on transfected cells. The results are summarized in the table 2 below. Different patterns of reactivity were observed: some sera reacted with the full length protein and intracellular domain while others also reacted with TRK1 and 2. As a whole, the four sera reacted with the intracellular domain of the FGFR3 protein in concordance with the ELISA method which used the intracellular domain of the protein.







TABLE 2







immunoreactivity of the serum of four patients with


anti-FGFR3 antibody by ELISA on HEK 293 cells


transfected with the full length FGFR3, the


intracytoplasmic domain, TRK1 or TRK2 subunit


of the intracellular domain of FGFR3.














FL-
intracellular





sera
FGFR3
domain
TRK1
TRK2







Patient 1
pos
pos
neg
neg



Patient 2
pos
pos
neg
neg



Patient 3
pos
pos
pos
pos



Patient 4
neg
pos
neg
neg



Control 1
neg
neg
neg
neg



Control 2
neg
neg
neg
neg



Control 3
neg
neg
neg
neg



Control 4
neg
neg
neg
neg







pos: positive.



neg: negative.






2.8. Discussion
The inventors have identified FGFRs and their associated GRB proteins as potential target of IgG autoantibodies in patients with sensory neuronopathy (SNN).
Elisa and protein arrays using a peptide from the intracellular kinase domain of FGFR3 (amino acids 399-806) allowed identification of a group of patients with SSN (most of them without known associated autoimmune context) harbouring anti-FGFR3 antibodies. Immunocytochemistry performed on cells transfected with the full length FGFR3, the intracellular domain (amino acids 397-806), TRK1 or TRK2 confirms that the FGFR3 anti-sera reacted with the tyrosine kinase domain, although some sera also reacted with TRK1 or TRK2.
As a whole, the FGFR3 antibody associated neuropathy follows the criteria of probable SNN in 82% of cases according to published criteria in patients with available information (Camdessanche J. P. et al., Brain 2009; 132:1723-1733), the others but one having sensory neurons involvement on clinical evaluation. In some patients, the sensory neuron disorder may present as small fibre neuropathy or trigeminal nerve neuropathy. Interestingly one patient for whom the diagnosis hesitated between CIDP and SNN and another one presenting with distal sensory neuropathy can be reallocated to a diagnosis of SNN because of the presence of FGFR3 antibody.
SNN may occur with lupus or Sjögren syndrome but the neuropathy frequently appears months or years before the autoimmune disorder becomes apparent while many cases of SNN never develop any autoimmune context even after a very protracted course. It is not established that when present the autoimmune disease is responsible for the neuropathy and most probably their co-occurrence results from the association of two autonomous autoimmune disorders. In this study, 47% of patients with FGFR3 antibodies have an autoimmune context including lupus or lupus anticoagulant, Sjögren syndrome, sclerodermy or unclassified autoimmune disorder. One patient had HIV infection. In the others, there was no known autoimmune context. Interestingly, in one of them a nerve biopsy disclosed vasculitis in the epinerium confirming that the disorder involves inflammatory mechanisms.
Therefore detection of FGFR3 antibodies early in the course of the neuropathy leads to ascribe the neuropathy to an otherwise undetected autoimmune disorder. There are to date no known biomarkers for non paraneoplastic SNN. The availability of such a diagnostic tool allows the identification of a group of patients who are currently considered as having an idiopathic disease and who can now be candidate to receive an immunological treatment.

## Claims

1. A method for treating a patient suffering from an acquired sensory neuronopathy who shows immunoreactivity toward a protein of the tyrosine kinase receptor family chosen from FGFR1, FGFR2 or FGFR3 and/or toward a GRB10 protein, which method comprises
a) detecting in a biological sample of the patient immunoreactivity toward a protein of the tyrosine kinase receptor family chosen from FGFR1, FGFR2 or FGFR3 and/or toward a GRB10 protein; and
b) administering to the patient at least an immunosuppressant and/or immunomodulator compound(s), or a pharmaceutical composition comprising said compound(s).

2. The method of claim 1, wherein the immunosuppressant compound is chosen from the group consisting of tacrolimus (CAS number 104987-11-3), cyclosporine (CAS number 59865-13-3), methotrexate (CAS number 59-05-2), glucocorticoids, cyclophosphamide (CAS number 50-18-0), azathioprine (CAS number 446-86-6), mycophenolate mofetil (CAS number 24280-93-1), anti-CD20 antibodies and fingolimod (CAS number 162359-55-9).

3. The method of claim 1, wherein the protein of the tyrosine kinase receptor family is chosen from the group consisting of i) FGFR3, ii) a protein which comprises or consists of the intracellular kinase domain of FGFR3, iii) a protein which comprises or consists of the TRK1 subunit of the intracellular kinase domain of FGFR3, and iv) a protein which comprises or consists of the TRK2 subunit of the intracellular kinase domain of FGFR3.

4. The method of claim 1, wherein the proteins toward which immunoreactivity is detected are i) a protein chosen from the group consisting of FGFR3, a protein which comprises or consists of the intracellular kinase domain of FGFR3, a protein which comprises or consists of the TRK1 subunit of the intracellular kinase domain of FGFR3, and a protein which comprises or consists of the TRK2 subunit of the intracellular kinase domain of FGFR3, and ii) GRB10.

5. An analytical method comprising
i) obtaining a biological sample from a patient having an acquired sensory neuronopathy and
ii) detecting, in the biological sample, antibodies toward a protein of the tyrosine kinase receptor family chosen from FGFR1, FGFR2 or FGFR3 and/or toward a GRB10 protein.