Methods and kits for diagnosing and / or treating peripheral neuropathy

Abstract

The present invention relates to a method and / or kit for diagnosing whether an individual suffers from peripheral neuropathy pain. The method comprises detecting advillin in a biological sample by detecting an immune complex formed by advillin and a monoclonal antibody that specifically binds thereto. The present invention also encompasses a method for treating an individual suffering from peripheral neuropathy pain.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 638,338, filed on March 5, 2018, which is hereby incorporated by reference in its entirety. Background of the Invention Technical Field

[0004] The present invention generally relates to the technical field of peripheral neuropathy (or peripheral neuropathy); in particular, to the technical field of diagnosis and treatment of peripheral neuropathy.

[0005] Prior Art

[0006] An individual with peripheral neuropathy will present with one or more peripheral nerve conditions. Peripheral nerves are tissues that extend outward from the brain and spinal cord beyond the central nervous system to provide a two-way communication network. They serve as conduits for impulses from the brain and spinal cord to the rest of the body; for example, motor neurons carry messages that direct movement. Peripheral nerves can also transmit sensory information to the brain that is collected by specific receptors. In simple terms, peripheral nerves provide a connection between the brain, body, and environment, and coordinate the relationship between the brain and the outside world of an organism.

[0007] There are many different peripheral neuropathies. Some are very common, while others are very rare. The causes of some peripheral neuropathies are well understood, while others are completely unknown. Many of these neuropathies have been classified into specific syndromes. Each syndrome is associated with its own combination of clinical symptoms and signs, prognosis, and treatment options. The importance of this is that a specific patient can be matched to a syndrome that corresponds to his or her clinical condition. Figure 1 Generally, such matching enables the physician to select a course of treatment and guide the patient as to prognosis. Often, identification of a specific syndrome alerts the physician to another medical condition that is associated with the patient's peripheral neuropathy, requires a specific course of treatment, and can predict its own prognosis. Therefore, being able to make a correct and accurate diagnosis is extremely important in the management of patients with peripheral neuropathy. However, making the correct diagnosis is not easy. In the past, such diagnoses relied heavily on analysis of the patient's symptoms and a close physical examination. Even more troubling is that many peripheral neuropathy syndromes have not yet been fully identified.

[0008] In view of the above, there is an urgent need in the relevant field for a novel marker that can help diagnose an individual suffering from peripheral neuropathy. Summary of the invention

[0009] The present invention is based, at least in part, on the unexpected discovery by the inventors that advillin, a sensory neuron-specific protein, can be used as a biomarker for diagnosing peripheral neuropathy. Accordingly, human monoclonal antibodies for detecting advillin can be used in methods and kits for detecting advillin in a biological sample, wherein the biological sample is derived from an individual suspected of suffering from peripheral neuropathy; and methods for diagnosing the individual.

[0010] Accordingly, the first aspect of the present invention is to provide a monoclonal antibody that can specifically bind to an epitope of advillin.

[0011] According to an embodiment of the present invention, the monoclonal antibody is produced by the fusion tumor YCC-S1.

[0012] According to an embodiment of the present invention, the monoclonal antibody is an IgG or an IgM.

[0013] A second aspect of the present invention is a method for diagnosing whether an individual suffers from peripheral neuropathy pain. The diagnosis is based on a biological sample of the individual, and the method comprises the following steps:

[0014] detecting a complex formed by advillin in the biological sample and the monoclonal antibody of the present invention in an immunological test;

[0015] Wherein formation of the complex indicates that the subject suffers from peripheral neuropathy pain.

[0016] According to an embodiment of the present invention, the monoclonal antibody is an IgG or an IgM.

[0017] According to an embodiment of the present invention, the biological sample is cerebrospinal fluid, peritoneal fluid, blood, serum, plasma or tissue biopsy.

[0018] According to an embodiment of the present invention, the peripheral neuropathy pain is caused by damage to sensory neurons, especially sensory neurons that can be identified by specific neural markers, such as sensory neurons that can specifically bind to isolectin B4, calcitonin gene-related peptide (CGRP), substance P or neurofilament 200.

[0019] According to an embodiment of the present invention, the immunological test is a western blot analysis, an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunohistochemistry (IHC) test or an immunocytochemistry (ICC) test.

[0020] A third aspect of the present invention is a method for treating a subject suffering from peripheral neuropathy pain. The method comprises:

[0021] obtaining a biological sample from the individual;

[0022] In an immunoassay, the biological sample is mixed with the monoclonal antibody of the present invention, so that advillin in the biological sample reacts with the monoclonal antibody to form a complex; and

[0023] An effective amount of a therapeutic agent is administered to the subject to improve the peripheral neuropathy pain, wherein the therapeutic agent is an analgesic, an anti-epileptic drug or an antidepressant.

[0024] According to an embodiment of the present invention, in step (a), the biological sample may be cerebrospinal fluid, peritoneal fluid, blood, serum, plasma or a biopsy tissue section.

[0025] According to an embodiment of the present invention, in step (b), the immunological test can be an immunoblot analysis, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunohistochemistry (IHC) assay or an immunocytochemistry (ICC) assay.

[0026] According to an embodiment of the present invention, the analgesic may be a nonsteroidal anti-inflammatory drug (NSAID), an opioid or oxycodone. Examples of NSAIDs suitable for use in the methods of the present invention include, but are not limited to, aspirin, ibuprofen, dexibuprofen, naproxen, fenoprofen, dexketoprofen, flurbiprofen, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, acelofenac, prioxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, mefenamic acid, and fenamic acid. acid), meclofenamic acid, flufenamic acid, and tolfenamic acid.

[0027] According to an embodiment of the present invention, the antiepileptic drug may be gabapentin or pregabalin.

[0028] According to an embodiment of the present invention, the antidepressant may be amitriptyline, doxepin or nortriptyline.

[0029] The details and description of one or more embodiments of the present invention are set forth below. Other features and advantages of the present invention will become apparent after reviewing the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] This patent or application contains at least one drawing executed in color.

[0031] The present invention will become more apparent after referring to the following detailed description and accompanying drawings, in which:

[0032] Figure 1Immunoblotting analysis of advillin in CSF of EAE mice. (a) Representative immunoblotting analysis results of advillin protein amount in cerebrospinal fluid (CSF) of mice with experimental autoimmune encephalomyelitis (EAE). CSF samples were collected from EAE mice in recovery. Each lane was injected with 4 μl of CSF sample or 10 μg of wild-type (WT) spinal cord lysate as a positive control group. Albumin was used as a loading control. The numbers below the genotype indicate the EAE clinical score on the day of CSF collection. Myosin IIa is a protein that interacts with advillin. (b) Results of quantification of advillin expression in CSF samples. The dotted line represents the threshold amount (mean + 5 times standard deviation of negative samples) to define positive advillin expression.

[0033] Figure 2 Advillin in CSF of oxaliplatin (Oxa) treated mice. (a) Representative immunoblot results for detection of advillin protein in CSF of oxaliplatin treated mice. Each lane was injected with 5 μl of CSF sample or 10 μg of WT spinal cord lysate as a positive control. Albumin was used as a loading control. The graph shows the time points of sample collection after oxaliplatin treatment. The delay of acute nocifensive response was used as an index of the severity of neuropathy pain. Myosin IIa is an advillin-interacting protein. SC WT is a lysate of spinal cord cells from WT mice. (b) Results of quantification of advillin expression in CSF samples. The dotted line indicates the threshold amount (mean + 5 times standard deviation of negative samples) to define positive advillin expression. Early stage is defined as day 1 to day 7 after oxaliplatin treatment, while late stage is defined as ≥ day 8.

[0034] Figure 3 Advillin protein in CSF of mice treated with oxaliplatin or complete Freund's adjuvant (CFA). Each lane was injected with 4 μl of CSF sample or 0.5 μg of dorsal root ganglion (DRG) cell lysate as a positive control. Albumin was used as a loading control. Advillin was detected only in CSF samples of mice treated with oxaliplatin, but not CFA.

[0035] Figure 4 Advillin protein in CSF of mice treated with chronic constriction injury (CCI) of the sciatic nerve. (a) Western blot analysis results for the amount of advillin protein in CSF of CCI mice. CSF samples were collected from mice subjected to CCI (7, 8, 9, and 10 days after surgery) and decompression. Each lane was injected with 6 μL of CSF sample or 0.5 μg of DRG lysate or 10 μg of WT spinal cord lysate (SC WT). WT DRG and WT SC served as positive controls. KO DRG served as negative control. Arrows indicate positive samples. (b) Results of quantification of advillin expression in CSF samples. The dotted line indicates the threshold amount (mean value of the initial sample + 5 times the standard deviation) to define positive advillin expression.

[0036] Figure 5 CCI induces an increase in advillin expression in the dorsal horn of the spinal cord. (a) The figure shows that the immunoreactivity of advillin (Avil) in the ipsilateral side (ipsi) dorsal horn of the spinal cord of CCI mice (day 9 after surgery) was significantly increased compared with the naïve and CCI mice (scale bar: 200 μm). (b) The figure shows the quantitative results, and the intensity of advillin immunoreactivity in the superficial dorsal horns was analyzed by one-way ANOVA. ****P<0.0001.

[0037] Figure 6 Human advillin monoclonal antibody can specifically recognize human advillin exogenously expressed in 293 cells. Human advillin clones were tagged with flag, and human advillin-flag (hAvil-flag) construct vectors or control group (flag) vectors were transfected into 293 cells. Lysates were detected with human advillin monoclonal antibody and flag antibody, respectively. The molecular weight of human advillin is predicted to be 92 kilodaltons (kDa), and the detected signal can correspond to the predicted size of hAvil-flag (about 100 kilodaltons). The signal above is inferred to be aggregated based on the detection of the signal recognized by the flag antibody.

[0038] Figure 7hAvil monoclonal antibody can be used to detect advillin expression in CSF of human patients treated with oxaliplatin. CSF samples were collected from patients treated with oxaliplatin chemotherapy. Each lane was injected with 30 μl of CSF sample (about 15 μg) or 2 μg of hAvil-flag transfected 293 cell lysate as a positive control. Albumin was used as a loading control for CSF. CSF numbers 73, 78, 79 and 80 are samples with significant signals (above the threshold). DETAILED DESCRIPTION

[0039] The detailed description and drawings provided below are intended to make the description of the embodiments of the present invention more detailed and complete, and are not intended to represent the only form for understanding or using the embodiments of the present invention. This specification describes the functions of the embodiments and the sequence of steps used to construct and operate these embodiments. However, other embodiments may also be used to achieve the same or equivalent functions and sequence of steps.

[0040] 1. Definition

[0041] For convenience, the specific terms used in the present invention are disclosed herein. Unless otherwise defined in this specification, the meanings of the scientific and technical terms used herein are the same as those understood and used by those with ordinary knowledge in the technical field to which the present invention belongs.

[0042] The term "antibody" or "antibodies" is used in the broadest sense, and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (as long as they exhibit the desired biological activity), that is, when in a complex mixture of proteins and / or other molecules, the antibody preferentially recognizes its target antigen and specifically binds to the antigen. According to the present invention, the antibody can be any type (e.g., IgG, IgM, IgD, IgE, IgA and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0043] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, and is not limited to being prepared by any particular method. In contrast to polyclonal antibodies, which generally include different antibodies against different epitopes, different monoclonal antibodies each target a single determinant position (i.e., epitope) on an antigen. A typical method of preparing a monoclonal antibody is by fusing a normally short-lived antibody-producing B cell with a fast-growing cell (e.g., an immortal cell), the resulting fused cell or hybridoma being able to replicate rapidly and from which a clone that can produce large quantities of the monoclonal antibody is produced. Alternatively, a monoclonal antibody may be prepared by recombinant DNA methods, wherein a portion of the heavy chain and / or light chain is identical or homologous to an antibody sequence derived from a particular species or belonging to a class or subclass of antibodies, and the remainder of the chain is identical or homologous to an antibody sequence derived from another species or belonging to another class or subclass of antibodies, as well as antibody fragments of such antibody sequences, as long as they exhibit the desired biological activity.

[0044] The term "subject" or "patient" refers to an animal that can be diagnosed by the present invention, including humans. Unless one gender is specifically indicated, the term "subject" or "patient" means both male and female. Accordingly, the term "subject" or "patient" includes any mammal that can benefit from the treatment methods of the present invention.

[0045] As used herein, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are identical or have a specific percentage of amino acid residues that are identical when the sequences are compared and arranged (i.e., sequence alignment) for maximum sequence correspondence. To determine the percentage of similarity, the sequences are arranged with the goal of obtaining the best alignment result (e.g., a gap can be introduced in a first amino acid sequence to achieve the best alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of similarity between two sequences is a function of the number of identical positions shared by the two sequences (i.e., % similarity = identical positions / total number of positions × 100). In certain embodiments, the two sequences are the same length. Therefore, 100% similarity means, for example, that when comparing 20 sequentially arranged amino acid residues in two different molecules, the 20 residues in the two different molecules are identical.

[0046] The singular forms "a," "an," and "the" herein include plural referents unless the context clearly dictates otherwise.

[0047] 1. Advillin monoclonal antibody

[0048] The present invention relates to anti-advillin monoclonal antibodies, fusion tumor cells for preparing the monoclonal antibodies, and their use in detecting advillin that may be present in a biological sample (such as a subject's cerebrospinal fluid (CSF), peritoneal fluid, blood, serum, plasma or a biopsy tissue section (such as skin)) to determine whether the subject of the biological sample suffers from peripheral neuropathy pain.

[0049] According to one aspect of the present invention, a monoclonal antibody that can specifically bind to human advillin is provided.

[0050] To prepare the desired monoclonal antibody, an experimental animal (e.g., mouse, rat, or rabbit) is first immunized with an appropriate dose of a synthetic peptide coupled to ovalbumin. In one embodiment, the synthetic peptide has an amino acid sequence KNQNQELPEDVNPAKKENYLSE (SEQ ID NO: 1) corresponding to the headpiece domain of human advillin. In another embodiment, the synthetic peptide has an amino acid sequence DGEPKYYPVEVLKGQNQEL (SEQ ID NO: 3) corresponding to the headpiece domain of mouse advillin. Generally speaking, when immunizing experimental animals, the adjuvant and antigen solution should be mixed evenly before administration. Examples of the adjuvant of the present invention include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and aluminum hydroxide adjuvant. Immunization can usually be performed by injecting the antigen intravenously, subcutaneously, intraperitoneally or intramuscularly, and the immunization time interval is not particularly limited. Immunization can be performed over a period of several days to several weeks, preferably within 2 to 3 weeks, and administered 1 to 10 times, preferably 2 to 5 times. Once the absorbance value of the antibody titer reaches 2 or higher (due to the immune effect), the experimental animal is left to stand for 2 to 6 months, preferably 4 to 6 months, and more preferably 6 months, until the antibody titer absorbance value is reduced to 0.05-1, preferably 0.05-0.5, and more preferably 0.05.

[0051] Then, multiple re-immunizations are performed, preferably 2 to 5 times of repeated stimulation of the experimental animals over a period of several weeks. Within a few days after the final immunization, preferably within 3 to 5 days, the spleen cells and regional lymph nodes of the experimental animals are removed. In addition, blood samples of the experimental animals must be taken regularly after the start of immunization to separate the serum by centrifugation. The antibody titer in the obtained serum is measured by appropriate detection methods, including, but not limited to, immunoblot analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC) test or immunocytochemistry (ICC) test. In a preferred embodiment, the antibody titer is detected by ELISA. Then, animals that produce high antibody titers to advillin are subjected to final immunization.

[0052] Antibody-producing cells are prepared from spleen cells and regional lymph nodes or their analogs from the immunized animals mentioned above. In the process of manufacturing antibody-producing cells, it is necessary to remove tissue fragments and red blood cells as much as possible. Commercial erythrocyte removers can be used to achieve this purpose. In addition, ammonium chloride and Tris buffer can also be used.

[0053] The antibody-producing cells thus prepared are immediately fused with immortal cells (e.g., myeloma cells) to produce hybridoma cells, which can continue to proliferate semi-eternally and produce antibodies at the same time. Common cell lines derived from animals (e.g., mice) can be used to achieve this purpose. The cell lines that can be used in the present invention should not survive in HAT selective medium (components include hypoxanthine, thymidine, and aminopterin), but when the cells are fused with cells that can produce antibodies, they should be able to grow in the medium. Examples of myeloma cells include, but are not limited to, mouse myeloma cell lines (e.g., myeloma FO cells) and human myeloma cell lines (e.g., Karpas 707H).

[0054] Cell fusion is usually performed by mixing spleen cells or lymph node cells with a commercially available myeloma cell in the presence of a cell fusion promoter (e.g., polyethylene glycol (PEG), with an average molecular weight of 200 to 20,000 Daltons or its analogs). Alternatively, cell fusion can be performed in a commercially available cell fusion device using electric current stimulation (e.g., electroporation). After cell fusion, the cells are diluted and cultured in HAT medium. For the purpose of preparing monoclonal antibodies, aminopterin in the culture medium blocks the de novo pathway. Therefore, since unfused myeloma cells cannot prepare nucleotides through de novo synthesis or salvage pathway, these cells will die. Since unfused B cells have a short lifespan, these cells will also die. In this way, only the fused cells of B cells and myeloma (i.e., specific fusion tumors) will survive. These cells will produce antibodies (a characteristic of B cells) and are immortal (a characteristic of myeloma cells). Next, the culture medium is diluted into a microtiter plate to a level where each well contains only one cell. Next, the supernatant in each well is collected and tested for the presence of advillin antibody titers. ELISA, EIA or RIA can be used as a confirmation method. Once the antibody-positive culture wells are confirmed, the cells are then cultured in an HT culture medium (without aminopterin). After a period of culture, the antibody titer in the culture supernatant needs to be confirmed again. The cells finally selected are then cloned to obtain single cells. A cloned strain that is highly specific to advillin is selected and proliferated to a certain extent to establish a fusion tumor.

[0055] According to a preferred embodiment of the present invention, a hybridoma was selected and named "YCC-S1". The selected hybridoma can produce monoclonal antibodies, and the monoclonal antibodies can specifically bind to human advillin exogenously expressed in 293T cells.

[0056] The monoclonal antibodies prepared by the above-mentioned fusion tumor can be separated or prepared by any known method. For example, the fusion tumor can be cultured in a medium with a low serum concentration, and the supernatant of the culture medium can be collected to obtain the antibody. Alternatively, the fusion tumor can be injected into the abdominal cavity of an animal, and then the ascites of the animal can be collected to prepare the antibody. The antibody can be purified or separated by affinity column, gel filtration chromatography, ion exchange resin or similar methods. In addition, the above-mentioned known methods can also be appropriately selected or used in combination to obtain antibodies.

[0057] According to one embodiment of the present invention, the monoclonal antibody produced by the selected clone can specifically bind to human advillin. This monoclonal antibody is an IgG, referred to herein as "hAvil". The hAvil of the present invention has been confirmed to have a heavy chain and a light chain. Each heavy chain or light chain includes a variable region composed of complementary-determination region (CDR) 1, CDR2 and CDR3. The hAvil of the present invention includes a heavy chain variable region, which includes amino acid sequences with sequence numbers: 22, 24 and 26; and a light chain variable region, which includes amino acid sequences with sequence numbers: 30, 32 and 34.

[0058] Alternatively, anti-advillin monoclonal antibodies can be prepared by DNA gene selection. Based on the amino acid sequence of hAvil, the DNA encoding hAvil can be easily isolated and sequenced using conventional procedures (e.g., oligonucleotide probes that can specifically bind to the heavy and light chain genes encoding monoclonal antibodies). A preferred source of DNA is to use these fusion tumor cell lines (e.g., YCC-S1 fusion tumor cell lines). Once isolated, the DNA can be constructed into an expression vector, which can then be transfected into a host cell (e.g., Escherichia coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins), thereby preparing the desired monoclonal antibody in the recombinant host cell.

[0059] According to an embodiment of the present invention, the nucleic acid encoding the heavy chain variable region of the anti-advillin monoclonal antibody includes polynucleotide sequences with sequence numbers 6, 8 and 10, which are respectively used to encode the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain. The nucleic acid encoding the light chain variable region of the anti-advillin monoclonal antibody includes polynucleotide sequences with sequence numbers 14, 16 and 18, which are respectively used to encode the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain.

[0060] The above-mentioned monoclonal antibodies and DNA encoding such antibodies can be used to prepare chimeric antibodies, humanized antibodies and / or antibody fragments derived therefrom.

[0061] 2. Methods and / or kits for diagnosing peripheral neuropathy pain

[0062] The present invention also encompasses a method for detecting and / or diagnosing whether a subject suffers from peripheral neuropathy pain.

[0063] According to certain embodiments of the present invention, advillin is present in the cerebrospinal fluid of experimental autoimmune encephalomyelitis (EAE) mice, which can simulate multiple sclerosis and show severe peripheral neuropathy associated with IB4+ sensory neurons (Wang et al., 2017; Scientific Reports 7: 42304). According to other embodiments of the present invention, advillin is also present in oxaliplatin-treated mice, which can simulate the peripheral neuropathy pain associated with IB4+ sensory neurons caused by chemotherapeutic agents. According to further embodiments, advillin is also present in mice treated with chronic compression of the sciatic nerve, which can simulate local neuropathy associated with IB4+ sensory neurons (Casals-Diaz et al., 2009; Exp Neurol 217: 84-95). Therefore, advillin can be used as a biomarker for diagnosing whether an individual has peripheral neuropathy pain.

[0064] Accordingly, the present invention also provides a method for diagnosing whether an individual suffers from peripheral neuropathy pain. The diagnosis is based on a biological sample taken from the individual, and the method comprises: detecting a complex formed by advillin in the biological sample and the monoclonal antibody of the present invention in an immunological test; wherein the formation of the complex indicates that the individual suffers from peripheral neuropathy pain.

[0065] According to one embodiment of the present invention, advillin can be detected by reacting the monoclonal antibody with a biological sample to form a complex, wherein the immunoassay includes, but is not limited to, immunoblotting analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC) assay or immunocytochemistry (ICC) assay. The measurement result can be used to indicate or determine whether the individual from whom the biological sample was collected suffers from peripheral neuropathy pain.

[0066] To achieve this purpose, a biological sample is taken from an individual suspected of suffering from peripheral neuropathy pain. The biological sample can be a whole blood sample, a plasma sample, a serum sample, an ascites (or peritoneal fluid) or a cerebrospinal fluid (CSF) sample, any of which is purified or filtered, or a skin biopsy. For early detection considerations, it is preferred to use blood, serum, plasma, ascites, CSF or skin biopsy samples. Then, the above biological sample is reacted with the monoclonal antibody of the present invention. Conventional ELISA or dot-blot analysis can be performed to measure peripheral neuropathy pain or measure the amount of advillin in the biological sample.

[0067] According to another embodiment of the present invention, advillin can be detected using an anti-advillin monoclonal antibody in a detection kit or as a reagent.

[0068] The inventors therefore designed an advillin detection kit that can measure the amount of advillin in a biological sample with high sensitivity. The kit of the present invention comprises at least the monoclonal antibody of the present invention; at least one agent suitable for detecting the binding of hAvil with advillin in the biological sample of the individual; and instructions for use associated with the kit for indicating how to use the kit.

[0069] The biological sample of the present invention includes, but is not limited to, a whole blood sample, a serum sample, a plasma sample, a CSF sample, a peritoneal fluid sample (or ascites) and its purified or filtered form, or a skin biopsy. The components included in the kit are: a container; the monoclonal antibody hAvil; a reagent for detecting a biological sample; and an instruction placed in the container and used to indicate how to use the monoclonal antibody to detect advillin in a biological sample. The instruction can be in the form of a pamphlet, a CD, a VCD, a DVD or a software application. The kit can further include a negative control for indicating the normal amount of advillin in a subject.

[0070] 3. Methods for treating peripheral neuropathy pain

[0071] The present invention also encompasses a method for treating an individual suffering from peripheral neuropathy pain. As described above, advillin can be used as a biomarker for diagnosing whether an individual suffers from peripheral neuropathy pain. Accordingly, once the individual is diagnosed with peripheral neuropathy pain, treatment and / or therapeutic measures can be administered to the individual. The method comprises the following steps,

[0072] obtaining a biological sample from the individual;

[0073] In an immunoassay, the biological sample is mixed with the monoclonal antibody of the present invention, so that advillin in the biological sample reacts with the monoclonal antibody to form a complex of advillin and the monoclonal antibody; and

[0074] An effective amount of a therapeutic agent is administered to the subject to improve the peripheral neuropathy pain, wherein the therapeutic agent is an analgesic, an anti-epileptic drug or an antidepressant.

[0075] According to an embodiment of the present invention, in step (a), the biological sample can be a whole blood sample, a plasma sample, a serum sample, an ascites (or peritoneal fluid) or a cerebrospinal fluid (CSF) sample, any of the purified or filtered forms thereof, or a skin biopsy. In terms of early detection, it is preferred to use blood, serum, plasma, ascites, CSF or skin biopsy samples.

[0076] In step (b), the biological sample of step (a) is uniformly mixed with the monoclonal antibody of the present invention to react, and the formation of the complex between advillin and the monoclonal antibody of the present invention in the biological sample is confirmed by conventional ELISA or dot blot analysis.

[0077] In step (c), an effective amount of a therapeutic agent is administered to the subject to improve the peripheral neuropathy pain. The therapeutic agent may be an analgesic, an antiepileptic or an antidepressant. Examples of analgesics suitable for use in the method of the present invention include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, dexibuprofen, naproxen, fenoprofen, dexketoprofen, flurbiprofen, cloxaprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, decefenacin, aceclofenac, piloxicam, meloxicam, tenoxicam, droxaxicam, lornoxicam, isoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid and tolfenamic acid; opioids, such as tramadol; oxycodone, such as oxycontin. Examples of anti-epileptic drugs suitable for use in the methods of the present invention include, but are not limited to, gabapentin and pregabalin. Examples of anti-depressants suitable for use in the methods of the present invention include, but are not limited to, amitriptyline, doxepin, and nortriptyline.

[0078] The present invention will be more specifically described with reference to the following embodiments, which are for illustrative purposes rather than for restrictive purposes.

[0079] Example

[0080] Materials and Methods

[0081] Experimental animals

[0082] All procedures followed the Guide for the Care and Use of Laboratory Animals (National Academies Press, Washington, DC). The advillin+ / cre mouse strain was a gift from Dr. Fan Wang (Duke University Medical Center, Durham, NC). This study used advillincre / cre mice as advillin knockout mice, which replaced the second exon of advillin by the Cre cassette, and homozygous gene-inserted mice did not express any advillin mRNA (Hasegawa H et al., J Neurosci (2007) 27 (52): 14404-14414; Zhou X, et al., Proc Natl Acad Sci USA (2007) 107 (20): 9424-9429).

[0083] For the behavioral studies, advillin+ / cre mice were backcrossed with C57BL / 6J mice for 10 generations to establish a congenic strain. The wild-type mice (WT or Avil+ / +) and advillin knockout mice (advillincre / cre or Avil- / -) used in the behavioral studies were the offspring of heterozygous (Avil+ / -) matings.

[0084] For immunoblot analysis of CSF samples, C57BL / 6J and BALB / cByJNarl mice were subjected to EAE induction and oxaliplatin treatment. Adult mice used in all studies were 8 to 12 weeks old unless otherwise stated.

[0085] Experimental Autoimmune Encephalitis (EAE) Model

[0086] For the EAE-inducing part, myelin oligodendrocyte glycoprotein peptide (35-55) (product name: MOG35-55, MDBio) was emulsified with an equal amount of Freund's complete adjuvant (CFA, Sigma-Aldrich, USA) in PBS. The MOG / CFA emulsion (MOG35-55 100 micrograms per mouse and CFA 400 micrograms per mouse) was subcutaneously injected into the hind limbs of mice, followed by intraperitoneal (ip) injection of 200 nanograms of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) on the day of immunization and 2 days later. Clinical scores were assessed every day and on the day of behavioral testing, pathological examination or CSF collection. The clinical scores can be referred to Wang IC et al. (Sci Rep (2017) 7:42304) with slight modifications: 0, no clinical signs; 0.5, partial paralysis of the tail; 1, paralysis of the tail; 2, ataxia of the lower body; 3, paraparesis of one or both hind limbs; 4, paraplegia of one or both hind limbs; 5, paraplegia of one or both hind limbs and incomplete paralysis of the forelimbs; 6, death.

[0087] Collection of cerebrospinal fluid (CSF)

[0088] Mice were anesthetized with urethane (13% w / v in saline, 1.5-2 mg / g, ip), the skin of the neck was shaved, and the mice were mounted on a stereotaxic system (Stoelting, Wood Dale, IL, USA). The skin below the occipital bone was incised longitudinally, followed by blunt dissection with forceps to separate the subcutaneous tissue and muscle. A pulled glass capillary was used to puncture the cisterna magna down to the dura mater. Approximately 6 μl of CSF was collected from each mouse and immediately placed in a test tube and frozen on dry ice and then transferred to a -80°C freezer. All CSF samples used for immunoblotting analysis showed no obvious visible blood contamination.

[0089] Preparation of rabbit anti-mouse advillin polyclonal antibody

[0090] A peptide "DGEPKYYPVEVLKGQNQEL" (SEQ ID NO: 3) corresponding to the bridle domain of mouse advillin was synthesized and coupled to ovalbumin. The synthetic peptide was administered to rabbits to stimulate the production of advillin polyclonal antibodies. Serum was collected and purified by column coupling with the corresponding antigen. For the immunostained skin tissue section, the advillin antibody was absorbed with 0.5% (wt / vol) acetone powder of advillin-deficient mouse liver to minimize nonspecific binding.

[0091] Immunoblotting analysis

[0092] The primary antibodies used for immunoblotting analysis were rabbit anti-mouse advillin polyclonal antibody (1 μg / mL), mouse anti-human advillin monoclonal antibody, and rabbit anti-albumin antibody (1:1000, both from GeneTex, San Antonio, TX, USA). Mouse DRG, mouse spinal cord, and human 293T cells (ATCC CRL-3216) were collected and proteins were extracted with radioimmunoprecipitation assay buffer (RIPA buffer) supplemented with protease inhibitors (Roche, Switzerland). Proteins in cerebrospinal fluid (CSF) samples or cell lysates were separated by SDS-PAGE and transferred to PVDF membranes using a transfer cell (Mini Trans-Blot Electrophoretic Transfer Cell) (Bio-rad, CA, USA). The membrane was blocked with 5% milk (in TBST, 1×TBS containing 0.1% Tween 20) (Sigma-Aldrich) for 1.5 hours at room temperature and incubated with the primary antibody for 1.5 hours at room temperature. The membrane was then washed three times with TBST for 10 minutes each time and incubated with the appropriate secondary antibodies (1:10,000, GeneTex): goat anti-rabbit IgG (H+L) horseradish peroxidase (HRP) and goat anti-mouse IgG (H+L) HRP. All antibodies were diluted in blocking solution. The signal was detected using enhanced chemiluminescence (ECL) from the Immobilon ECL Kit (Millipore, MA, USA) and the BioSpectrum Auto Imaging System (UVP, CA, USA). The advillin in CSF samples was detected using a chemiluminescence enhancer kit (SuperSignal West Femto ECL kit) (Thermo Fisher Scientific, PA, USA).

[0093] Plasmid construction

[0094] The full-length cDNA of human advillin (GenBank accession no. NM_006576) obtained from human placenta cDNA was cloned into a vector (pGEM-T easy vector) (Promega, USA). PCR cloning was performed using a forward primer 5'-gagggatccatgcctctgaccagtgccttca-3' (SEQ ID NO: 36) and a reverse primer 5'-cgctctagacttgctttagaaaagcccctt-3' (SEQ ID NO: 37) with a stop codon. The cDNA of advillin was subcloned into an expression vector (p3xFLAG-CMV14) (Sigma).

[0095] Cell culture and transfection

[0096] HEK293T cells were maintained in DMEM (Invitrogen, CA, USA) supplemented with penicillin (100 units per ml) / streptomycin (100 μg per ml) (Invitrogen) and 10% (vol / vol) fetal bovine serum (FBS) (Invitrogen). For transfection of plasmids, when the confluence of HEK293T cells was >90%, transfection was performed with transfection reagent (lipofectamine 2000) (Invitrogen). The primary culture of DRG neurons was performed according to the steps described by Cheng CM, et al. (Nat Protoc (2010) 5(4):714-724). Briefly, whole DRGs of mice (6 to 12 weeks old) were excised and collected by a two-step digestion: first, 0.125% collagenase type I (Sigma) (in HBSS) (Invitrogen) for 60 minutes at 37°C, followed by 0.125% trypsin (Invitrogen) (in HBSS) for 20 minutes at 37°C. Completely digested DRG cells were rinsed with DMEM (Invitrogen) containing 10% (vol / vol) FBS, then triturated and seeded on coverslips coated with poly-L-lysine (PLL) and laminin (both purchased from Sigma). Cells were cultured in DMEM containing 10% (vol / vol) FBS and penicillin (100 units per ml) / streptomycin (100 μg per ml) at 5% CO2 and 37°C.

[0097] Pain behavior

[0098] Cold allodynia was assessed by a cooling plate test with an innocuous temperature (Toyama S, et al. (2014) Anesthesiology 120(2):459-473). The temperature of the cooling plate (35100, Ugo Basile, Milan, Italy) was set to 15°C and allowed to stabilize for 5 minutes. The mice were placed on the cooling plate and placed in a 3.5 cm high transparent plexiglass chamber to prevent the mice from standing. The behavior was recorded for 150 seconds. During the recording period, the acute pain behavior was evaluated by counting the number of flicking, shaking, licking, flinching, guarding, lifting the forepaws and licking the hindpaws, and body jumping or shaking. In addition, the delay to the first acute pain response can be confirmed. The behavior of mice administered oxaliplatin (15 mg / kg, ip) was recorded before drug injection (as baseline) and on days 2, 4, and 12 after drug administration. This experiment was blinded, meaning that the experimenter who performed the calculation of acute pain behavior was blinded to the genotype of the mice.

[0099] Mouse model of CCI decompression

[0100] The chronic constriction injury (CCI) mouse model can be found in Tseng TJ, et al. (2008) Exp Neurol 204(2):574-582; Lorenz JE, et al. (2014) Antioxid Redox Signal 21(10):1504-1515. Briefly, mice were anesthetized with 2% isoflurane. The left sciatic nerve was exposed to the mid-thigh and loosely ligated with 6-0 silk thread at three locations with a distance of 1 mm. The incised skin was then sutured with 5-0 silk thread. After the 14th day of sciatic nerve compression, the mice were anesthetized and all loose ligatures were carefully removed for decompression.

[0101] Immunohistochemistry and quantitative imaging

[0102] Adult mice (8 to 12 weeks old) with or without CCI surgery were anesthetized with urethane (13% weight / volume in saline, 1.5-2 mg / g, ip), perfused cardiovascularly with saline, followed by 4% tris(formaldehyde) (Sigma) fixative in 0.1 volume molar concentration PBS (pH = 7.4). Spinal cords were collected, post-fixed overnight at 4°C, cryoprotected in 30% sucrose solution overnight, and frozen in OCT (Leica, Germany) in preparation for cryosectioning. Tissues prepared for cryosectioning were stored at -80°C until use. Sections were rinsed with TBST (TBS containing 0.1% Tween 20) and exposed to blocking solution (5% BSA, 0.1% tritonX-100 in TBS) for 1 hour at room temperature. The primary antibody goat anti-CGRP (Serotec, UK), advillin, and the secondary antibody conjugated with a fluoride series fluorescent dye (AlexaFluor) (Invitrogen) were diluted in blocking solution. The sections were exposed to the primary antibody overnight at 4°C, rinsed three times with TBST, and then exposed to the secondary antibody for 90 minutes at room temperature. After rinsing, the sections were embedded in mounting medium (Vectashield mounting medium) (Vector Laboratories, CA, USA). Immunohistochemical images were obtained using a confocal microscope (LSM 700confocal microscope) (Carl Zeiss, Germany). Software (ImageJ) was used to measure the advillin immunoreactivity in the dorsal horn of the spinal cord. After removing the background value, the ratio of the ipsilateral dorsal horn to the contralateral dorsal horn was calculated.

[0103] Preparation of human advillin monoclonal antibody

[0104] To prepare the immunogen, the amino acid "cysteine" was added to the N-terminus of the synthetic peptide "KNQNQELPEDVNPAKKENYLSE" (SEQ ID NO: 1) corresponding to the bridle domain of human advillin, and it was coupled to the carrier ovalbumin. Balb / c mice were immunized and given a primary injection (0.1 mg of antigen and complete adjuvant) and a boost injection (0.1 mg of antigen and incomplete adjuvant). After the polyclonal serum was verified, a final boost (0.05 mg of antigen) was performed, and then fusion tumor cells were prepared, subcloned by limiting dilution, and screened by ELISA using another synthetic peptide "LKNQNQELPEDVNPAKKENYLSEQD" (SEQ ID NO: 2). The verified clones can be analyzed by immunoblotting and confirmed with an expression plasmid for human advillin.

[0105] The components and nucleic acid and amino acid sequences of the human advillin monoclonal antibody of the present invention are summarized in Tables 1 and 2.

[0106] Table 1 Heavy chain sequences of anti-advillin antibodies

[0107]

[0108] Table 2 Light chain sequences of anti-advillin antibodies

[0109]

[0110] Example 1 Advillin as a biomarker for detecting peripheral neuropathy

[0111] 1.1 Advillin is present in the CSF of EAE mice

[0112] The inventors have found in previous studies that during axonal regeneration, advillin is located in the growth cones of neurites, and in cultured DRG neurons, after neurite retraction, the newly formed focal-adhesion protein complex associated with advillin is usually "shed-off". Therefore, it is speculated that nerve damage (i.e., peripheral neuropathy) on isolectin B4+ (IB4+) sensory axons may cause newly formed focal-adhesion proteins associated with advillin to shed from the growth cones, and eventually advillin can be detected in CSF or plasma.

[0113] To prove the above speculation, a mature and developed multiple sclerosis mouse model was used for experiments, wherein the mouse model can show severe peripheral neuropathy associated with IB4+ sensory neurons, namely the experimental autoimmune encephalomyelitis (EAE) mouse model. According to the steps described in the "Materials and Methods" section, the EAE model was established, and the symptoms caused by EAE in mice were evaluated by the degree of paralysis of the tail and limbs as a clinical score of disease severity and reflecting the degree of nerve damage. CSF of naive mice and EAE mice were collected respectively, and the presence of advillin was analyzed by immunoblotting.

[0114] As expected, advillin protein was detected in the CSF of EAE mice during the recovery period, which was defined as after day 30 of the EAE model, when a high proportion of mice had a clinical score of ≥ 3. However, advillin was barely detectable in initially healthy mice ( Figure 1 ). The quantitative results are summarized in Table 3.

[0115] Table 3 Analysis of advillin expressed in CSF of naïve and EAE mice by quantitative immunoblotting

[0116]

[0117] The experimental results of this example confirm the above speculation, that is, peripheral neuropathy can cause the new advillin-related punctate adhesion proteins to fall off from the growth cones, and eventually advillin can be detected in the CSF of the damaged individual.

[0118] 1.2 Advillin is present in CSF of animals with chemotherapy-induced cold tenderness

[0119] To simulate chemotherapy-induced neuropathy pain, test animals were given a single dose of oxaliplatin (15 mg / kg, ip) to induce cold allodynia, and then the latency to the first acute pain sign was recorded. CSF was collected from both naive mice and mice with oxaliplatin-induced cold allodynia and analyzed for the presence of advillin by immunoblotting.

[0120] Similar to the findings in Example 1.1, advillin could be detected in mice that were given a cold stimulus 2 or 4 days after treatment with oxaliplatin and that exhibited a short latency (<65 seconds) to the cold stimulus, but could not be detected in mice that exhibited a long latency (<65 seconds) to the cold stimulus. Figure 2 ).

[0121] 1.3Advillin is not present in the CSF of animals with inflammatory pain induced by complete Freund's adjuvant (CFA)

[0122] To confirm that advillin is a biomarker specific for peripheral neuropathy pain, complete Freund's adjuvant (CFA) was injected into the test animals to induce inflammatory pain. CSF of each animal was collected and analyzed for the presence of advillin. For comparison purposes, CSF of animals treated with oxaliplatin (e.g., animals of Example 1.2) was also collected and analyzed.

[0123] like Figure 3 As shown, advillin could not be detected in the CSF of animals with CFA-induced inflammatory pain.

[0124] 1.4 Advillin is present in the CSF of animals treated with chronic constriction injury of the sciatic nerve

[0125] In this example, mice that can simulate severe neuropathy pain associated with local nerve damage caused by compression of the sciatic nerve are used to verify whether advillin works on this type of pain. In brief, the sciatic nerve of the mouse is surgically ligated (or compressed) to produce chronic constriction injury (CCI). Decompression surgery is performed on the 14th day after CCI, i.e., the ligature on the sciatic nerve is removed. CSF of CCI mice and control group mice is collected and analyzed for the presence of advillin by immunoblotting.

[0126] like Figure 4 As shown in the experimental data of , advillin can be detected in CCI mice, but not in CCI-decompressed mice.

[0127] In addition, the distribution of advillin in naïve, CCI, and CCI-decompressed mice was also confirmed. Figure 5 As shown, sciatic nerve compression significantly increased advillin expression in the dorsal horn of the affected side on day 9, and it returned to the normal basal amount of naive mice 2 weeks after decompression.

[0128] Summarizing the experimental data of Examples 1.1, 1.2, 1.3 and 1.4, advillin can be used as a specific biomarker for diagnosing peripheral neuropathy pain.

[0129] Example 2 Confirmation of the specificity of human advillin monoclonal antibody

[0130] In order to achieve the purpose of specific detection of human samples, human advillin monoclonal antibody (herein referred to as "hAvil") was prepared according to the steps described in the "Materials and Methods" section. The specificity of hAvil was verified by binding to an exogenously expressed human advillin clone ( Figure 6 ). Further, hAvil can specifically detect advillin in human CSF collected from patients treated with oxaliplatin ( Figure 7 ).

[0131] The above experimental data confirm that hAvil is highly specific against human advillin and can be used for clinical detection of advillin in the CSF of individuals suffering from peripheral neuropathy pain.

[0132] Although the above embodiments disclose specific embodiments of the present invention, those with ordinary knowledge in the art to which the present invention belongs can make various changes and modifications thereto. The above description, examples and experimental data provide a complete description of the structure and use of the present invention as an exemplary embodiment. Although the above description of various embodiments of the present invention has been described to a certain extent, or with reference to one or more individual embodiments, those with ordinary knowledge in the art to which the present invention belongs can still make many modifications to the disclosed embodiments without departing from the spirit and scope of the present invention.

Claims

1. A monoclonal antibody that specifically binds to an epitope of advillin, comprising a heavy chain variable region, comprising a heavy chain complementary determining region 1, a heavy chain complementary determining region 2 and a heavy chain complementary determining region 3, wherein the heavy chain complementary determining region 1, the heavy chain complementary determining region 2 and the heavy chain complementary determining region 3 consist of the amino acid sequences of SEQ ID NOs: 22, 24 and 26, respectively; and A light chain variable region, comprising a light chain complementary determining region 1, a light chain complementary determining region 2 and a light chain complementary determining region 3, wherein the light chain complementary determining region 1, the light chain complementary determining region 2 and the light chain complementary determining region 3 are composed of the amino acid sequences of sequence numbers: 30, 32 and 34, respectively.

2. The monoclonal antibody according to claim 1, wherein The heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence of sequence numbers: 6, 8 and 10; and The light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence of sequence numbers: 14, 16 and 18.

3. The monoclonal antibody of claim 1, wherein the antibody is an IgG or an IgM.

4. A kit comprising the monoclonal antibody according to claim 1, and instructions for use associated with the kit for indicating how to use the kit.

5. Use of the monoclonal antibody according to claim 1 in preparing a kit for detecting whether advillin is expressed in an individual suffering from peripheral neuropathy pain, wherein the kit is based on a biological sample of the individual, In an immunological test, the expression of advillin in the individual is determined by the complex formed by advillin in the biological sample and the monoclonal antibody.

6. The use according to claim 5, wherein the biological sample is cerebrospinal fluid, peritoneal fluid, blood, serum, plasma or biopsy tissue.

7. The use according to claim 5, wherein the peripheral neuropathy pain is caused by damage to sensory neurons.

8. The use according to claim 7, wherein the sensory neuron specifically binds to isolectin B4, calcitonin gene-based peptide, substance P or neurofilament 200.

9. The method of claim 5, wherein the immunological assay is an immunoblot analysis, an enzyme-linked immunosorbent assay, a radioimmunoassay, an immunohistochemical assay or an immunocytochemical assay.

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