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WO2002002620A2 - Acide nucleique, polypeptide de crmp-5 et utilisations de ceux-ci - Google Patents

Acide nucleique, polypeptide de crmp-5 et utilisations de ceux-ci Download PDF

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Publication number
WO2002002620A2
WO2002002620A2 PCT/US2001/020507 US0120507W WO0202620A2 WO 2002002620 A2 WO2002002620 A2 WO 2002002620A2 US 0120507 W US0120507 W US 0120507W WO 0202620 A2 WO0202620 A2 WO 0202620A2
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Prior art keywords
crmp
polypeptide
nucleic acid
biological sample
antibody
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PCT/US2001/020507
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English (en)
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WO2002002620A3 (fr
Inventor
Vanda A. Lennon
Zhiya Yu
Thomas J. Kryzer
Guy E. Griesmann
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Mayo Foundation For Medical Education And Research
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Publication of WO2002002620A3 publication Critical patent/WO2002002620A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • This invention relates generally to paraneoplastic neurological syndromes and cancer, and more particularly, to the involvement of CRMP-5 polypeptides in paraneoplastic syndromes and cancer.
  • the present invention provides for CRMP-5 nucleic acids and polypeptides and uses thereof.
  • Neoplasms expressing these organ-restricted proteins as immunogens include thymomas and carcinomas of the lung, ovary, breast and testis. It is clear that both host and tumor factors are determinants of paraneoplastic immune responses, but molecular determinants of immunogenicity in individual tumors are unknown.
  • a number of onconeural antigens have been defined in the nucleus, cytoplasm and plasma membrane of neurons and muscle by their specific reactivity with autoantibodies in patients' sera.
  • Antibodies reactive with cation channels and neurotransmitter receptors residing in synaptic plasma membranes have the potential to interfere with neural transmission in the peripheral nervous system and the central nervous system.
  • Autoantibodies directed at intracellular antigens are unlikely to be either neuropathogenic or inhibitory to tumor growth, but those of the IgG class reflect helper T cell activation.
  • IgG autoantibodies of neuronal nuclear and certain cytoplasmic specificities are usually accompanied by inflammatory lesions in the neuraxis and a distant occult neoplasm with limited metastasis. These autoantibodies lack demonstrable pathogenicity, but are surrogate markers of T lymphocyte activation, and predict specific cancers.
  • the present invention describes a novel IgG and its antigen.
  • the novel IgG is an anti-neuronal autoantibody marker of limited small-cell lung carcinoma and thymoma in individuals with unusual multifocal neurological disorders.
  • the antigen is a novel isoform of the neuronal collapsin response-mediator protein (CRMP) family and was designated CRMP-5.
  • CRMP-5 is highly expressed in neurons throughout the adult nervous system and is expressed in small-cell lung carcinomas and neuroblastoma.
  • the present invention provides for an isolated nucleic acid selected from the following: (a) an isolated nucleic acid having the nucleotide sequence of SEQ ID NO:l; (b) a fragment of the nucleic acid of (a), wherein the fragment is: (i) a fragment consisting of nucleotide 1 through at least nucleotide 544 of SEQ ID NO:l; (ii) a fragment of at least 70 nucleotides in length from nucleotide 544 through nucleotide 1695 of SEQ ID NO: 1 ; or (iii) a fragment of at least 70 nucleotides in length comprising nucleotide 544 of SEQ ID NO:l within the at least 70 nucleotide fragment; (c) a nucleic acid that is at least 92% identical to (a) or (b); and (d) a nucleic acid complementary to (a), (b) or (c).
  • the above-described nucleic acid may encode a polypeptide having the amino
  • vectors containing an above-described nucleic acid and host cells containing those vectors.
  • a vector may include regulatory elements that are necessary for expression and that are operably linked to the nucleic acid.
  • the present invention further provides host cells containing those expression vectors. Representative host cells include bacterial, yeast, insect and animal cells.
  • a method of producing a CRMP-5 polypeptide comprising the steps of: (a) culturing host cells containing the above- described expression vector under conditions permissive for expression of the nucleic acid; and (b) recovering polypeptides resulting from the expression of the nucleic acid.
  • the invention provides for a method of detecting the presence or absence of an anti-CRMP-5 autoantibody in an individual's biological sample.
  • This method comprises the steps of: (a) contacting the biological sample with a CRMP-5 polypeptide or fragment thereof; and (b) detecting the presence or absence of binding of the CRMP-5 polypeptide to the anti-CRMP-5 autoantibody in the biological sample.
  • the presence of the anti-CRMP-5 autoantibody in the biological sample is associated with paraneoplastic autoimmunity in the individual, which is, in turn, typically associated with a neoplasm, such as small-cell lung carcinoma, neuroblastoma and thymoma, in the individual.
  • Representative biological samples include blood, serum and cerebrospinal fluid.
  • an antibody i.e., polyclonal or monoclonal having specific binding affinity for a CRMP-5 polypeptide.
  • a monoclonal antibody having specific binding affinity for a CRMP-5 polypeptide may be produced by a hybridoma cell line such as CR1 or CR3.
  • Another aspect of the invention is a method of detecting the presence or absence of a CRMP-5 polypeptide in a biological sample from an individual. This method comprises the steps of: (a) contacting the biological sample with an antibody having specific binding affinity for a CRMP-5 polypeptide; and (b) detecting binding of the antibody to the biological sample.
  • Binding is indicative of the presence of the CRMP-5 polypeptide in the biological sample, and the presence of the CRMP-5 polypeptide in the biological sample is indicative of a neoplasm.
  • the neoplasm is small-cell lung carcinoma, thymoma or neuroblastoma.
  • Representative examples of biological samples are blood, serum, cerebrospinal fluid, pleural fluid, ascites, saliva, sputum, urine, stool or solid tissues.
  • the present invention also provides for a kit containing a CRMP-5 polypeptide.
  • the kit may further comprise a monoclonal antibody having specific binding affinity for a CRMP-5 polypeptide.
  • Figure 1 is the cDNA and predicted polypeptide sequence of human CRMP-5.
  • the underlined amino acids represent the polypeptide fragments generated by proteolytic cleavage of the polypeptide isolated from human brain.
  • Figure 2 is a restriction map of a CRMP-5 nucleic acid.
  • A Aspl; B, BamBI; H, HinaTII; N, Ncol; P, Pvull; R, Real; S, Sacl.
  • the human 62-kD antigen was immunochemically identified and purified, and the oncological and neurological correlations between the novel autoantibody and the novel antigen are reported herein. From partial amino acid sequence data from the purified antigen, a cDNA encoding the antigen was cloned and its tumor expression and antigenicity demonstrated.
  • the previously undescribed onconeural antigen is related to the collapsin-response-mediator protein (CRMP) family involved in axon guidance in the nervous system and was designated CRMP-5.
  • CRMP-5 defines CRMP-5 as a novel onconeuronal protein that elicits spontaneous immune responses in patients with small- cell lung carcinoma and thymoma.
  • the invention also defines IgG autoantibodies specific for CRMP-5 and provides examples of their potential clinical applications.
  • CRMP-5 onconeuronal antigen was assigned to the collapsin response- mediator protein (CRMP) family (averaging 50% amino acid identity with the other CRMP family members, Table 1) rather than a dihydropyriminidase (DHPase; 57% identity with CRMP-5, Table 1), based on the following: CRMP-5 lacks one of four invariant histidines critical for DHPase enzymatic activity (i.e., CRMP-5 contains conserved histidine (H) residues at positions 68, 70 and 191 of SEQ ID NO:2 and an asparagine (N) instead of an H at position 247), and expression of CRMP-5 is restricted to the nervous system.
  • CRMP collapsin response- mediator protein
  • the invention features an isolated nucleic acid encoding a human CRMP-5 polypeptide, and the complement thereof.
  • nucleic acid refers to RNA or DNA.
  • the invention also features CRMP-5 polypeptides.
  • CRMP-5" refers to a novel polypeptide of the neuronal collapsin response-mediator protein (CRMP) family. CRMP-5 is expressed in neurons of the adult central and peripheral nervous system, in small-cell lung carcinomas and neuroblastomas.
  • isolated refers to (i) a nucleic acid sequence encoding part or all of the human CRMP-5 polypeptide, but free of coding sequences that normally flank one or both sides of the nucleic acid sequences encoding CRMP-5 in the human genome; or (ii) a nucleic acid inco ⁇ orated into a vector or into the genomic DNA of an organism such that the resulting molecule is not identical to any naturally-occurring vector or genomic DNA.
  • isolated refers to a polypeptide that constitutes the major component in a mixture of components, e.g., 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more by weight.
  • Isolated polypeptides are typically obtained by purification from an organism that makes the polypeptide, although chemical synthesis is also feasible. Methods of polypeptide purification include, for example, chromatography or immunoaffinity techniques.
  • FIG. 1 An example of a nucleotide sequence encoding a human CRMP-5 polypeptide is shown in Figure 1 (SEQ ID NO: 1), which is a 1695 bp cDNA having an open reading frame of 1695 nucleotides.
  • the predicted amino acid sequence of a human CRMP-5 is shown in SEQ LD NO: 2, and is 564 amino acids in length.
  • the invention includes fragments of the human CRMP-5 nucleic acid and polypeptide.
  • fragments of the invention refer to nucleic acids or polypeptides corresponding to less than the entire CRMP-5 sequence.
  • Nucleic acid fragments may include those fragments starting at nucleotide 1 and continuing through at least nucleotide 544 of SEQ ID NO:l; fragments of at least 70 nucleotides in length between nucleotide 544 and nucleotide 1695 (inclusive) of SEQ ID NO:l; or those fragments of at least 70 nucleotides in length that include nucleotide 544 of SEQ ID NO:l within the 70 nucleotide or greater fragment.
  • Fragments provided by the invention include, for example, fragments from nucleotides 551-650, 1001-1100 or 1401-1500 of SEQ ID NO: 1.
  • the invention further provides novel fragments that are less than 70 nucleotides and that have utility as hybridization probes or PCR primers (e.g., nucleotides 738-774, 1115-1133 or 1543-1557 of SEQ LD NOT).
  • Additional fragments of the invention include, for example, nucleotides 26-775, 775-929, 1147-1652 or 479-1408 of SEQ LD NO: 1. Such fragments may, for example, encode a CRMP-5 antigenic polypeptide fragment.
  • Figure 2 shows the relative position of various restriction enzyme sites within the human CRMP-5 nucleic acid sequence that, by way of example, define positions, which, in various combinations, can be used to generate useful nucleic acid fragments.
  • any nucleic acid fragment can be generated by known means (e.g., restriction enzyme digestion, the polymerase chain reaction) and, if so desired, expressed to produce the corresponding polypeptide fragment.
  • the human CRMP-5 polypeptide can be cleaved (e.g., proteolytically) to directly generate polypeptide fragments. Representative examples of CRMP-5 fragments generated by proteolytic cleavage are shown underlined in Figure 1.
  • a human CRMP-5 nucleic acid or nucleic acid fragment may have a sequence that deviates from that shown in SEQ ID NOT or fragment thereof.
  • a nucleic acid sequence can have at least 92 percent (%) sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid sequence can have at least 95% sequence identity or at least 99% sequence identity to SEQ ID NOT.
  • Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid or polypeptide sequences, dividing the number of matched positions by the total number of aligned nucleotides or amino acids, respectively, and multiplying by 100.
  • a matched position refers to a position in which identical nucleotides or amino acids occur at the same position in aligned sequences.
  • the total number of aligned nucleotides or amino acids refers to the niinimum number of CRMP-5 nucleotides or amino acids, as disclosed in SEQ ID NOT or 2, respectively, that are necessary to align the second sequence, and does not include alignment (e.g., forced alignment) with non-CRMP-5 sequences, such as those fused to CRMP-5.
  • the total number of aligned nucleotides or amino acids may correspond to the entire CRMP-5 sequence (i.e., 1695 nucleotides or 560 amino acids) or may correspond to fragments of the full-length CRMP-5 sequence as defined herein. Sequences can be aligned using the Sequence Analysis Software Package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Sequence (i.e., pairwise) analysis of human CRMP-5 was performed with GCG Gap using the algorithm of Needleman & Wunsch (J.
  • a nucleic acid encoding a human CRMP-5 polypeptide may be obtained from, for example, a cDNA library made from a human small-cell lung carcinoma cell line, or can be obtained by other means, including, but not limited to, the polymerase chain reaction (PCR).
  • PCR refers to a procedure or technique in which target nucleic acids are amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
  • Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995.
  • sequence information from the ends of the region of interest or beyond is employed to design ohgonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • Representative ohgonucleotide primers for CRMP family members are shown in Table 2 and SEQ ID NOS:5-28 Human CRMP-5 nucleic acids can be detected by, for example, a variety of hybridization techniques. Hybridization typically involves Southern or Northern blotting (see, for example, sections 9.37-9.52 of Sambrook et al., 1989, "Molecular Cloning, A Laboratory Manual", 2 nd Edition, Cold Spring Harbor Press, Plainview; NY).
  • Oligonucleotides should hybridize under high stringency conditions to a human CRMP-5 nucleic acid (e.g., DNA or RNA), or the complement thereof.
  • High stringency conditions typically include the use of low ionic strength and high temperature washes, for example 0.015 M NaCl/0.0015 M sodium citrate (0.1X SSC), 0.1% sodium dodecyl sulfate (SDS) at 65°C.
  • Denaturing agents such as formamide, can additionally be employed during high stringency hybridization (e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH
  • CRMPl-4 primer 1 and 2 were used for full length PCR; primer 3 and 4 were used for nest PCR.
  • CRMP-5 Forward and Reverse primers were used for cloning the full length cDNA from both human brain and SCC-9 cDNA pools; TK2 (F), and TK3 (R ) were degenerate primers corresponding to peptides VIPGGI and VRGVDRTP for initial CRMP-5 cDNA cloning. I.U.P.A.C.
  • the present invention further includes vectors containing the human CRMP-5 nucleic acid of SEQ ID NOT (or the complement thereof), CRMP-5 nucleic acid fragments (or the complements thereof) and those nucleic acids having at least 92% sequence identity to SEQ ID NO or fragments generated therefrom (or the complements thereof).
  • Vectors of the invention may additionally comprise elements necessary for expression operably linked to a human CRMP-5 nucleic acid sequence.
  • elements necessary for expression include promoter sequences, and additionally may include regulatory elements, such as enhancer sequences, response elements or inducible elements that modulate expression of the human CRMP-5 nucleic acid sequence.
  • operably linked refers to positioning of a promoter and/or other regulatory element(s) in a construct relative to the human CRMP-5 nucleic acid sequences in such a way as to direct or regulate expression of the CRMP-5 nucleic acid.
  • Such constructs are commercially available (e.g., expression vectors) and/or produced by recombinant DNA technology methods routine in the art.
  • the choice of expression systems depends upon several factors, including, but not limited to, replication efficiency, selectability, inducibility, targeting, the level of expression desired, ease of recovery and the ability of the host to perform post- translational modifications.
  • host or "host cell” is meant to include not only prokaryotes, such as E. coli, but also eukaryotes, such as yeast, insect, plant and animal cells.
  • Animal cells include, for example, COS cells and HeLa cells.
  • a host cell can be transformed or transfected with a DNA molecule (e.g., a vector) using any of the techniques commonly known to those of ordinary skill in this art, such as calcium phosphate or lithium acetate precipitation, electroporation, lipofection and particle bombardment.
  • Host cells containing a vector of the present invention may be used for pu ⁇ oses such as propagating the vector, producing human CRMP-5 nucleic acid (e.g., DNA, RNA, antisense RNA) or expressing the human CRMP-5 polypeptide or fragments thereof.
  • methods of producing CRMP-5 polypeptides are provided. Methods of producing CRMP-5 polypeptides include, but are not limited to, culturing host cells containing a CRMP-5 expression vector under conditions permissive for expression of CRMP-5, and recovering the CRMP-5 polypeptides. Methods of culturing bacteria and recovering expressed polypeptides are well known to those of ordinary skill in this art.
  • nucleic acids of the present invention may be detected by methods such as Southern or Northern blot analysis (i.e., hybridization), PCR or in situ hybridization analysis.
  • CRMP-5 proteins are typically detected by sodium dodecyl sulphate (SDS)-polyacrylamide gel eleetrophoresis followed by Coomassie Blue-staining or Western blot analysis, using antibodies (monoclonal or polyclonal) that have specific binding affinity for a human CRMP-5 polypeptide.
  • the present invention provides for methods of using CRMP-5 polypeptides to detect anti-CRMP-5 autoantibodies in an individual.
  • the individual is typically displaying abnormal neurological symptoms of unknown origin.
  • the method of the invention is based on an association between the abnormal neurological symptoms, the presence of the anti-CRMP-5 autoantibody and the presence of neoplasms (e.g., neuroendocrine neoplasms, such as small-cell lung carcinoma, thymoma and neuroblastoma).
  • neoplasms e.g., neuroendocrine neoplasms, such as small-cell lung carcinoma, thymoma and neuroblastoma.
  • polypeptides and polypeptide fragments used for detection of anti-CRMP-5 autoantibodies in the methods of the present invention are specifically reactive with anti- CRMP-5 autoantibodies.
  • Polypeptides generally correspond to at least one epitopic site that is characteristic of a CRMP-5 protein.
  • Epitopes of the CRMP-5 polypeptide that are pertinent to T-cell activation and suppression are also provided by the invention.
  • Computer algorithms are available for predicting binding epitopes, e.g., MHC-I and MHC-II binding epitopes. See, for example, http ://bimas .dcrt.nih.
  • the term "characteristic" in this context means that the epitopic site allows immunologic detection of anti-CRMP-5 antibody or antigenic CRMP-5 polypeptides in sera with reasonable assurance. Usually, it is desirable that the epitopic site be antigenically distinct from other members of the CRMP family.
  • CMRP-5 polypeptides may be obtained from cells (e.g., transfected host cells) expressing a CRMP-5 nucleic acid, or the polypeptides may be synthetic.
  • a DNA molecule encoding a CRMP-5 polypeptide of fragment thereof may itself be natural or synthetic, with natural genes obtainable from human tissues by conventional techniques.
  • the CRMP-5 polypeptides are obtained in a substantially pure form.
  • the polypeptides may be purified by routine protein purification methods, including affinity chromatography (e.g., as described herein), or immunosorbant affinity column.
  • CRMP-5 polypeptides of the present invention may be used with or without modification for the detection of CRMP-5.
  • polypeptides are labeled by either covalently or non-covalently combining the polypeptide with a second substance that provides for detectable signal.
  • labels and conjugation techniques are known in the art and are reported extensively in both the scientific and patent literature.
  • Some of the labels include radioisotopes, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles and the like.
  • CRMP-5 polypeptides prepared as described above can be used in various immunological techniques for detecting anti-CRMP-5 autoantibodies in serum samples, such as from blood and cerebrospinal fluid. Depending on the nature of the sample, both immunoassays and immunocytochemical staining techniques may be used. Enzyme- linked immunosorbent assays (ELISA), Western blot and radioimmunoassays are routine methods in the art and may be used to detect the presence of anti-CRMP-5 autoantibodies in sera. Further provided by the present invention are kits containing CRMP-5 polypeptides. The kit may further include a second substance that provides for detectable signal. A kit typically also includes directions for using the CRMP-5 polypeptide and/or practicing the method (i.e., detecting anti-CRMP-5 autoantibody).
  • the present invention also provides a method of detecting CRMP-5 polypeptides in a biological sample from an individual.
  • the method describes an association between the presence of CRMP-5 and neoplasms (e.g., small-cell lung carcinomas and neuroblastomas) in the individuals screened.
  • neoplasms e.g., small-cell lung carcinomas and neuroblastomas
  • This test is most widely applicable to those individuals who do not present with abnormal neurological symptoms, but are suspected to have a new or recurrent neoplasm, e.g., small-cell lung carcinoma.
  • Detection of a protein is typically performed using an antibody, and the present invention also provides for an antibody, preferably a monoclonal antibody with specific binding affinity for
  • CRMP-5 polypeptides are examples of CRMP-5 polypeptides.
  • CRMP-5 are defined as antibodies that bind CRMP-5 but that do not bind, for example, either CRMP-2 or CRMP-3.
  • antibody refers to whole antibodies of any class, i.e., IgG, IgA, IgM or any other known class, and includes portions or fragments of whole antibodies (e.g., Fab or (Fab) 2 fragments) having the desired affinity, an engineered single chain Fv molecule, or a chimeric molecule, e.g., an antibody that contains the binding specificity of one antibody (e.g., of marine origin) and the remaining portions of another antibody (e.g., of human origin).
  • Hybridomas that produce monoclonal antibodies having specific binding affinity for CRMP-5 have been deposited with ATCC (10801 University Boulevard., Manassas, VA 20110-2209) and were assigned the following Accession number(s):
  • Anti-CRMP-5 antibodies of the present invention may be used with or without modification for the detection of CRMP-5.
  • antibodies are labeled either directly or indirectly, and a wide variety of labels, including radioisotopes, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers and magnetic particles, and conjugation techniques are known and are reported extensively in both the scientific and patent literature.
  • Antibodies prepared as described above can be used in various immunological techniques for detecting CRMP-5 polypeptides in a biological sample.
  • a "biological sample”, as used herein, is generally a sample from an individual. Non-limiting examples of biological samples include blood, serum, or cerebrospinal fluid, pleural fluid, ascites, saliva, sputum, urine or stool. Additionally, solid tissues, for example, lymph node specimens, may be used, e.g., for intraoperative diagnosis. The use of antibodies in protein binding assays is well established. Depending on the nature of the sample, imrnunoassays (e.g., radioimmunoassays) and/or immunohistochemical/immunocytochemical staining techniques may be used.
  • imrnunoassays e.g., radioimmunoassays
  • immunohistochemical/immunocytochemical staining techniques may be used.
  • Liquid phase imrnunoassays or Western blot analysis can also be used to detect CRMP-5 in a biological sample.
  • enzyme-linked immunosorbent assays ELISA
  • Numerous competitive and noncompetitive protein-binding assays have been described in the scientific and patent literature, and a large number of such assays are commercially available.
  • An example of one such competitive assay for detecting the presence of, for instance, the CRMP-5 polypeptide in a biological sample such as blood comprises: contacting a CRMP-5 polypeptide (either labeled or unlabeled) with an anti- CRMP-5 antibody (either labeled or unlabeled) and the biological sample.
  • the CRMP-5 polypeptide may be, for example, attached to a solid surface.
  • the relative amount of CRMP-5 polypeptide in a biological sample can be determined.
  • the kit may also include CMRP- 5 polypeptides or fragments thereof to be used as binding controls or to generate a standardized quantitative curve.
  • the kit may further include a second substance that provides for detectable label.
  • a kit typically includes directions for using the anti- CRMP-5 antibody and/or practicing the method (i.e., detecting CRMP-5 polypeptides). Also provided by this invention is an antibody having specific binding affinity for
  • CRMP-5 conjugated to a detectable marker Suitable detectable markers include, but are not limited to, enzymes, radioisotopes, dyes and biotin. This invention further provides an antibody having specific binding affinity for CRMP-5 conjugated to an imaging agent. Suitable imaging agents include, but are not limited to, radioisotopes, such as P, Tc, In and 131 I.
  • compositions comprising CRMP-5 polypeptide, alone or conjugated to a detectable marker, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (e.g., oil/water) or various types of wetting agents.
  • the method comprises administering an effective amount of a pharmaceutical composition of a CRMP-5 polypeptide or a nucleic acid encoding the CRMP-5 polypeptide to the individual.
  • An effective amount is an amount or CRMP-5 polypeptide that deviates the individual's CRMP-5 -mediated immune response, thereby modulating a neurological disorder in the individual.
  • administering means a method of administering to the patient. Such methods are well known to those skilled in the art and include, but are not limited to, administration orally, intravenously, or parenterally.
  • Also provided by this invention is a method of imaging CRMP-5-expressing neoplastic cells in a patient.
  • the method comprises administering to the patient an effective amount of an antibody having specific binding affinity for CRMP-5, labeled with an imaging agent, for example, 32 P, 99 Tc, ⁇ n In or 131 I, to bind to a CRMP-5 antigen released from, or accessible in, neoplastic cells shed from the tumor, and detecting any complex so formed.
  • an effective amount of antibody or composition is any amount that is effective to image the neoplastic cells, for example, about 0.1 mCi to about 50.0 mCi.
  • an effective amount of the antibody may be an amount from about 0.01 mg to about 100 mg.
  • Suitable methods of administering the imaging agent are as described above. Methods of imaging are dependent upon the agent used and are well known to those of skill in this art.
  • One method comprises administering to the patient, or to isolated antigen presenting cells (APCs) from the patient, an effective amount of a vaccine to stimulate cytotoxic T-cells.
  • the vaccine is a pharmaceutical composition comprising the CRMP-5 polypeptide of the invention, or a nucleic acid encoding the CRMP-5 polypeptide.
  • the CRMP-5 polypeptide may be co-administered to the patient with an immunomodulatory or immunostimulatory molecule, or administered via at least one nucleic acid encoding a CRMP-5 polypeptide and an immunomodulatory/immunostimulatory molecule (e.g., a single nucleic acid may encode a CRMP-5 -immunomodulatory or CRMP-5 -immunostimulatory fusion protein).
  • suitable immunomodulatory or immunostimulatory molecules include, for example, cytokines (e.g., GM-CSF, IL-12, IL-10) or unmethylated CpG sequences.
  • An effective amount is an amount that effectively modulates or stimulates the patient's immune response such that the neoplastic cells are killed or their proliferation inhibited.
  • a method of enumerating or isolating CRMP-5 -specific T- lymphocytes in an individual may be used, for example, to monitor an individual's immune response or for immunotherapy using CRMP-5 antigen-specific cytotoxic T-cells.
  • the method comprises contacting a patient-derived biological sample containing lymphocytes with tetrameric soluble class I major histocompatibility complex
  • MHC bearing identical antigenic CRMP-5 polypeptide fragments.
  • Linker molecules such as avidin and biotin, are used to produce the CRMP-5-MHC tetrameric complex and can subsequently be labeled with an indicator molecule, such that those T-cells that recognize the CRMP-5-MHC tetrameric complex are enumerated or isolated (e.g., using FACS analysis). See, for example, Schwartz, RS, New England J. Med. , 339: 1076-8,
  • Buffers contained protease inhibitors (phenyhnethylsulfonyl fluoride, 2 mM; Pepstatin A, 0.1 ⁇ g/ml; and Aprotinin, 1 KJU/ml).
  • Human cerebral grey matter was homogenized (1 g/10 ml) in 10 mM N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer (pH 7.4), containing 1 mM MgCl , 1 mM ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).
  • protease inhibitors phenyhnethylsulfonyl fluoride, 2 mM; Pepstatin A, 0.1 ⁇ g/ml; and Aprotinin, 1 KJU/ml.
  • Human cerebral grey matter was homogenized (1 g/10
  • Polypeptides in the second precipitate were dissolved in phosphate-buffered saline (pH 7.4), dialyzed against 10 mM HEPES (pH 7.4) containing 500 mM NaCl, and applied sequentially to two affinity resins (Affi-gel 10, Bio-Rad Laboratories, Hercules, CA) coupled with IgGs (10 mg/ml) purified by protein G-agarose adso ⁇ tion from serum of a healthy subject or a reactive individual.
  • affinity resins Adfi-gel 10, Bio-Rad Laboratories, Hercules, CA
  • Polypeptides that bound to the second resin were eluted in 6 M urea containing 2% 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate) (CHAPS), concentrated by centrifugation dialysis using a Centricon-10 membrane (Millipore Co ⁇ oration, Bedford, MA), and analyzed by gel eleetrophoresis. Protein bands visualized by Coomassie blue staining were excised and digested with lysyl-endopeptidase (Wako Chemicals USA, Richmond, VA).
  • CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate)
  • RNA extracted from the small-cell lung carcinoma line SCC-9 was reverse transcribed to provide first-strand complementary DNA (cDNA).
  • Degenerate ohgonucleotide primers encoding two peptide sequences obtained from the brain antigenic polypeptide (VTPGGI (SEQ ID NO:3) and VRGVDRTP (SEQ ID NO:4)) were used to amplify DNA encoding a CRMP-5 antigen by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a 1.5 Kb cDNA product was ligated into a TA-cloning vector (Invitrogen, Carlsbad, CA), and used to transform competent E. coli cells (INV ⁇ F 1 ; Invitrogen). DNA sequences of 23 selected clones were compared to sequences in the GenBank database.
  • CRMP-2 cDNA (GenBank accession Number D78013) was also cloned from the brain library using CRMP-2-specific primers (Table 2; SEQ ID NO: 13- 16). Each cDNA was cloned by insertion into a TA-cloning vector. cDNAs encoding full-length CRMP-5 and CRMP-2 polypeptides and two CRMP-5 fragments (amino acid residues 1-181 or 57-351) were inserted into the pTrcHis expression vector (Invitrogen). Recombinant polypeptide synthesis was induced in transformed E.
  • both anti-CRMP-5 and anti-amphiphysin autoantibodies bound to cytoplasmic components of neurons (synapse-rich regions and some somata) and to enteric neural elements. Both antibodies spared Purkinje somata, gut smooth muscle and mucosa, and kidney tissue. Immunoperoxidase staining revealed an abundance of both the novel CRMP-5 antigen and amphiphysin in the neuropil and some neuronal bodies throughout the brain, spinal cord and autonomic ganglia; midbrain synaptic staining was most intense. In dorsal root ganglion, the novel CRMP-5 antigen was more intense in small neurons than in large neurons. Amphiphysin immunoreactivity also was more intense in small neurons than large, by comparison with staining by the paraneoplastic autoantibody ANNA-1 (or "anti-Hu"). Normal IgG did not bind to any elements.
  • the novel anti-CRMP-5 autoantibody bound to an aqueous brain protein of apparent molecular weight 62-kD under standard Western blot conditions.
  • Initial analysis of Western blots on the same preparation of aqueous brain protein suggested that the anti- CRMP-5 autoantibody bound to a protein having a molecular weight of 66 kD.
  • Binding by the anti-CRMP-5 autoantibody was readily distinguished from immunoreactive bands yielded by ANNA-1, amphiphysin and other previously characterized paraneoplastic IgG autoantibodies.
  • the novel anti-CRMP-5 autoantibody bound an apparent tetrameric form (-264 kD) of the CRMP-5 antigen.
  • two- dimensional gel eleetrophoresis and Western blotting data show that native CRMP-5 exists in phosphorylated and non-phosphorylated forms.
  • the aqueous immunoreactive antigen isolated from human brain yielded eight polypeptides after proteolytic cleavage (underlined in Figure 1). All had sequence homology to dihydropyrimidinase (DHPase) and to CRMP family members.
  • DHPase dihydropyrimidinase
  • a full- length cDNA encoding the brain antigen was cloned from a human adult brain cDNA pool and assigned GenBank Accession No. AF157634.
  • the predicted polypeptide ( Figure 1) contains all eight peptide sequences obtained from cleavage of the purified aqueous brain polypeptide, and has 50% amino acid identity to the previously known four human CRMP family members, and 57% identity to human DHPase (Table 1).
  • CRMP-5 lacked one of four invariant histidine residues critical for DHPase activity, the antigen was assigned to a novel CRMP family designated CRMP-5.
  • the predicted size of the CRMP-5 antigen based on the amino acid sequence is 62 kD.
  • Example 5 Neurological and Oncological Findings in CRMP-5-Positive Individuals Clinical information was available for 102 of the 106 seropositive individuals.
  • ANNA-1 -positive patients 4 were found at autopsy.
  • other types of neoplasms e.g., carcinomas of colon, skin, breast, prostate and ureter, a B -lymphoma, and an unidentified cerebral mass.
  • the possible existence of an occult primary lung neoplasm was excluded by autopsy in only one individual. All 10 individuals who lacked documentation of a neoplasm had a history of tobacco use, and all but one were known to have had relentless neurological progression; 4 of those died without autopsy.
  • Thymoma was found in two women (aged 52 and 53) and two men (aged 39 and 40). Two of the four individuals with thymoma had my asthenia gravis, 1 had limbic encephalitis, and 1 had systemic lupus erythematosus.
  • B-lymphoma, 1 had an intracerebral mass.
  • Cerebral cortex 42 dementia (26); personality change (10); seizures (10); depression (9); confusion (8); psychosis (4); aphasia (1)
  • ⁇ Subacute chorea/athetosis (rarely documented with paraneoplastic autoimmunity) affected 3 men and 8 women. Four of these individuals had coinciding loss of smell and taste. Two men had recent-onset Parkinsonism.
  • Fifteen small-cell lung carcinoma cell lines were established in the Mayo Clinic's Neuroimmunology Laboratory (SCC-2, 4, 9, 15, 17, 18, 21, 24, 37, 58, 59, 81, 86, 110, 117 and six were obtained from the American Type Culture Collection (ATCC, Manassas, VA; NCI-H69, NCI-H128, NCI-H345, NCI-H146, NCI-H82, NCI-H209). The following human cell lines were obtained from the ATCC and used as controls:
  • IMR-32 (neuroblastoma), TE-671 (rhabdomyosarcoma), Jurkat (T lymphoma), HEK-293 (human embryonic kidney epithelium cells) and HeLa (cervical carcinoma).
  • RNA transcripts for CRMP-2, CRMP-4 and CRMP-5 were amplified by RT-PCR from a standard small-cell lung carcinoma line (SCC-9).
  • SCC-9 standard small-cell lung carcinoma line
  • CRMP-1, CRMP-2 CRMP-3, CRMP-4 and CRMP-5 transcripts were amplified from the human brain cDNA library
  • the CRMP-5 nucleic acid constructs and polypeptides provided by the invention allow for detection of anti-CRMP-5 IgG autoantibodies in an individual's sera.
  • the presence of the autoantibody correlates with small-cell lung carcinoma or thymoma (Table 3).
  • the frequency of detection of anti-CRMP-5 autoantibody is approximately 2 per 1,000 sera tested.
  • the CRMP-5 IgG was not encountered in research studies involving large numbers of healthy subjects or individuals with a variety of neurological disorders or neoplasms, with the exception of a single individual not exhibiting any paraneoplastic neurological manifestations out of 58 individuals previously diagnosed with small-cell lung carcinoma.
  • the invention also provides for methods to detect the CRMP-5 polypeptide using an anti-CRMP-5 antibody having specific binding affinity for CRMP- 5. Since only a small percentage of individuals with small-cell lung carcinomas present with paraneoplastic neurological symptoms, an anti-CRMP-5 monoclonal antibody may be used to screen the general population for early detection of neoplasias (e.g., small-cell lung carcinomas, neuroblastomas and thymomas) based on the presence or absence of CRMP-5 polypeptides. Monoclonal IgGs with specific binding affinity for CRMP-5 polypeptides (e.g., CR1, CR3) were generated by immunizing a rat with native CRMP-5 purified from human brain.
  • an anti-CRMP-5 monoclonal antibody may be used to screen the general population for early detection of neoplasias (e.g., small-cell lung carcinomas, neuroblastomas and thymomas) based on the presence or absence of CRMP-5 polypeptide

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Abstract

L'invention concerne des acides nucléiques et polypeptides de CRMP-5. Les polypeptides de CRMP-5 sont utiles pour détecter des auto-anticorps anti-CRMP-5 chez un sujet présentant des manifestations neurologiques paranéoplasiques. Une association a été établie entre les auro-anticorps anti-CRMP-5 et des néoplasmes. De plus, l'invention concerne des anticorps possédant une affinité de liaison spécifique pour CRMP-5. Les anticorps monoclonaux sont utiles pour détecter la présence de polypeptides de CRMP-5 chez des sujets, sur la base d'une association établie entre les polypeptides de CRMP-5 et des néoplasmes.
PCT/US2001/020507 2000-06-29 2001-06-28 Acide nucleique, polypeptide de crmp-5 et utilisations de ceux-ci WO2002002620A2 (fr)

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FR2853909A1 (fr) * 2003-04-16 2004-10-22 Galderma Res & Dev Genes du psoriasis
WO2008000512A2 (fr) * 2006-06-30 2008-01-03 Schwarz-Pharma Ag Procédé d'identification de modulateurs des crmp
EP3564670A1 (fr) * 2018-05-03 2019-11-06 Hospices Civils de Lyon Anticorps dirigés contre trim9 et/ou trim67 dans des syndromes neurologiques paranéoplasiques

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2853909A1 (fr) * 2003-04-16 2004-10-22 Galderma Res & Dev Genes du psoriasis
WO2004094660A2 (fr) * 2003-04-16 2004-11-04 Galderma Research & Development, S.N.C. Gene dpysl3 comme marqueur du psoriasis
WO2004094660A3 (fr) * 2003-04-16 2004-12-29 Galderma Res & Dev Gene dpysl3 comme marqueur du psoriasis
WO2008000512A2 (fr) * 2006-06-30 2008-01-03 Schwarz-Pharma Ag Procédé d'identification de modulateurs des crmp
WO2008000512A3 (fr) * 2006-06-30 2008-03-06 Sanol Arznei Schwarz Gmbh Procédé d'identification de modulateurs des crmp
EP3564670A1 (fr) * 2018-05-03 2019-11-06 Hospices Civils de Lyon Anticorps dirigés contre trim9 et/ou trim67 dans des syndromes neurologiques paranéoplasiques
WO2019211392A1 (fr) * 2018-05-03 2019-11-07 Hospices Civils De Lyon Anticorps contre le trim9 et/ou le trim67 dans des syndromes neurologiques paranéoplasiques

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