EP1230358A2 - N-cam homologe und dessen spleissvarianten - Google Patents

N-cam homologe und dessen spleissvarianten

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Publication number
EP1230358A2
EP1230358A2 EP00969784A EP00969784A EP1230358A2 EP 1230358 A2 EP1230358 A2 EP 1230358A2 EP 00969784 A EP00969784 A EP 00969784A EP 00969784 A EP00969784 A EP 00969784A EP 1230358 A2 EP1230358 A2 EP 1230358A2
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EP
European Patent Office
Prior art keywords
cam
nucleic acid
acid sequence
product
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00969784A
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English (en)
French (fr)
Inventor
Kinneret Savitzky
Jeanne Bernstein
Anat David
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Compugen Ltd
Original Assignee
Compugen Ltd
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Filing date
Publication date
Priority claimed from IL13245999A external-priority patent/IL132459A0/xx
Priority claimed from IL13493200A external-priority patent/IL134932A0/xx
Application filed by Compugen Ltd filed Critical Compugen Ltd
Publication of EP1230358A2 publication Critical patent/EP1230358A2/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above.
  • the present invention further concerns methods for screening for candidate activator or deactivators utilizing said amino acid sequences, as well as therapeutical and diagnostic utilization of said novel sequences.
  • Cell adhesion molecules are transmembrane glycoproteins with extracellular binding domains and cytoplasmic functional domains. Ligand binding to the extracellular domain initiates intracellular events through the cytoplastmic functional domain. These in turn cause a major behavioral and functional change in the cells.
  • CAMs Cell adhesion molecules
  • two complementary molecules are required, the adhesion molecule and its ligand, one on each side of the adhesion site.
  • the immunoglobulin-like super family of adhesion molecules is a large and diverse family of molecules so named because they have one or more
  • immunoglobulin-like domains Included within the group are molecules concerned with antigen recognition by an adhesion to lymphocytes. These include CD3, CD4 and CD8 which together recognize complexes of antigen peptide and the major histocompatibility complex on other cells, and lymphocyte function related antigens such as CD2 and others of the LFA group of molecules.
  • IAM intercellular adhesion molecules
  • NIC AM neural cell adhesion molecules
  • LI and TAG involved in organization and function of nerves.
  • Cell adhesion molecules are responsible for more than just adhesion of cells to one another and to their insoluble matrices. Additional functions include the ordering of cell sorting, migration and differentiation; organization of cell motility via the cytoskeleton; regulation of inter and intracellular signaling; and control gene transcription.
  • CAMs Cell adhesion molecules
  • CAMs are a subset of the immunoglobulin super family found in the nervous system of both vertebrates and invertebrates. They are usually surface membrane proteins with multiple Ig domains in their N-termini followed by several fibronectin-type III repeats and either a transmembrane intracellular domain or a glycophosphatidyl inositol- linked membrane anchor at the C-terminus.
  • NCAM neuronal cell adhesion molecule
  • CAM neuronal cell adhesion molecule
  • CAM is a member of the immunoglobulin super family and is strongly expressed in the nervous system.
  • CAM is found in three major forms of which two - NCAM- 140 and NCAM- 180 are transmembrane proteins, while the third - NCAM- 120 is attached to the membrane via a glycosylphosphatidyl inositol anchor.
  • soluble NCAM forms exist in the brain, cerebrospinal fluid and in the plasma. NCAM mediates cell adhesion through homophilic as well as heterophilic activities. Following NCAM binding, transmembrane signalling is believed to be achieved resulting in increased
  • NCAM By mediating cell adhesion to other cells and to the extracellular matrix, and by activating intracellular signal pathways, NCAM influences cell migration, neurite extension and possibly also formation of synapses in the brain. From some studies on NCAM knock-out mice, it has been deduced that NCAM is crucial for the formation of the olfactory bulb and the mossy fiber system in the hippocampus. In addition, NCAM is important for neuronal plasticity in the adult brain associated with learning and regeneration.
  • This protein may be a secreted adhesion-like molecule with anti-protease activity.
  • a mutated or partially deleted sequence results in Kallmann Syndrome which is a genetic disorder that associates hypogonadotropic hypogonadism and anosmia. In this disease, the normal embryonic migration of GNRH-synthesizing neurons from the olfactory placodes to the hypothalamic region as well as the axonal extension of olfactory neurons towards the forebrain are impaired.
  • NCAM is expressed also in a number of different tissues and cell types, beyond neurons, such as muscles and endocrine-originating tumors, and can be detected in sera of patients with small-cell lung cancer.
  • NCAM nuclear factor-binding protein
  • Cell adhesion molecules homolog (CAM-H) nucleic acid sequence - the sequence shown in SEQ ID NO: 1 or 7, sequences having at least 70% identity to said sequence and fragments of the above sequences being 20 b.p. long.
  • These sequences contain an N-terminal which has a homology to neural cell adhesion molecules containing several repeats of the Ig-like C2-domain which are immunoglobulin-like domains involved in protein-protein and protein- ligand interactions.
  • the N-terminal part of the novel sequences of the invention are homologues to Contactin/F3 -subgroup adhesion molecules; NB-2, Tag-1 (Axonin-1), Big-1, Big-2, and Contactin.
  • the novel sequences of the invention contain in their C-terminal neuropilin- like MAM domain.
  • Neuropilin is a calcium-independent cell adhesion molecule that functions during the formation of certain neuronal circuits. This protein binds to Semaphorin III and to the VEGF165 isoform of VEGF.
  • the MAM domain has been recognized as the extracellular region of functionally diverse proteins. All the protein have a modular receptor-like architecture consisting of a signal peptide, followed by a large N-terminal extracellular domain, a single transmembrane region and an intracellular region.
  • SEQ ID NOS: 1 to 6 are from Homo sapiens and SEQ ID NO: 7 is from mouse.
  • SEQ ID NO: 4 is in fact identical to SEQ ID NO: 1 - and was inserted under a different name for convenience reasons.
  • SEQ ID NO: 5 is an update of SEQ ID NO: 2 (i.e. a sequence where 3 nucleotides have been altered).
  • SEQ ID NO: 1 (and 4), 2 (and 5), 3 and 6 are in fact all splice variants of each other.
  • CAM-H does not necessarily signify that the protein coded by the above sequences has the same or even similar physiological activities to known CAM molecules and merely indicates that it shows sequence homology with two CAMs.
  • CAM homology product (CAM-H product) - also referred at times as the "CAM-H protein” or "CAM-H polypeptide” - is an amino acid coded by the nucleic acid sequences of SEQ ID NO: 1 to SEQ ID NO: 7.
  • the amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein.
  • An example of an CAM-H product is shown in SEQ ID NO: 8 to SEQ ID NO: 13 (coded by the amino acid sequences of SEQ ID NO: 1 to SEQ ID
  • the term also includes analogues of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments of this sequence having at least 10 amino acids.
  • Nucleic acid sequence a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
  • amino acid sequence - a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
  • “Fragments of CAM-H nucleic acid sequence” a continuous portion, preferably of about 20 nucleic acid sequences of the CAM-H nucleic acid sequence.
  • Conservative substitution - refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
  • Non-conservative substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.
  • “Chemically modified” - when referring to the product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art.
  • modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
  • Bioly active refers to the CAM-H product which has physiological, regulatory or biochemical functions on the same target sites which naturally occurring CAM-H effects, for example can bind to the same ligands as the CAM, for example ligands present in extracellular matrixes; can interact with the same ligands present on the surface cells with which CAMs interact etc.
  • "Immunologically active” defines the capability of a natural, recombinant or synthetic CAM-H product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • a biologically active fragment of CAM-H product denotes a fragment which retains some or all of the immunological properties of the CAM-H product, e.g can bind specific anti-CAM-H product antibodies or which can elicit an immune response which will generate such antibodies or cause proliferation of specific immune cells which produce CAM-H.
  • Optimal alignment is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using a ktup of 1 , default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from Mac Vector (TM), operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the "gap" of the first sequence).
  • TM Mac Vector
  • Having at least X% identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned.
  • 70% amino acid sequence identity means that 70% of the amino acids in two or more optimally aligned polypeptide sequences are identical.
  • isolated nucleic acid molecule having an CAM-H nucleic acid sequence is a nucleic acid molecule that includes the coding CAM-H nucleic acid sequence.
  • Said isolated nucleic acid molecule may include the CAM-H nucleic acid sequence as an independent insert; may include the CAM-H nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the CAM-H coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the CAM-H nucleic acid sequence may be in combination with non-coding sequences, e.g., introns or control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the CAM-H protein coding sequence is a heterologous.
  • Expression vector refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • “Deletion " - is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • “Insertion” or “addition " - is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
  • substitution - replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non- conservative.
  • Antibody refers to IgG, IgM, IgD, IgA, and IgG antibody.
  • the definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-CAM-H product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
  • the term “antibody” may also refer to antibodies against nucleic acid sequences obtained by cDNA vaccination.
  • Activator refers to a molecule which mimics the effect of the natural CAM-H product or at times even increases or prolongs the duration of the biological activity of said product, as compared to that induced by the natural product.
  • the mechanism may be by binding to the same moiety to which native CAM-H binds (for example on the extracellular matrix or to a protein present on the membrane of other cells) thus mimicking the activity of CAM-H; by prolonging the lifetime of the CAM-H, (for example by decrease of the rate of its degradation), by increasing the affinity of CAM-H to moieties which it binds (such as the extracellular moieties) etc.
  • Activators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which can bind to and activate the CAM-H product.
  • Deactivator or (“Inhibitor”) - refers to a molecule which modulates the activity of the CAM-H product in an opposite manner to that of the activator, by decreasing or shortening the duration of the biological activity of the CAM-H product. This may be done by blocking the binding of the CAM-H to the moiety for example to the ligand to which it binds by competitive or non-competitive inhibition, by causing rapid degradation of the CAM-H, etc. by inhibiting association of the CAM-H with the effectors which regulate its expression.
  • Deactivators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of said product.
  • “Treating a disease” - refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
  • Detection refers to a method of detection of a disease. This term may refer to detection of a predisposition to a disease.
  • Probe - the CAM-H nucleic acid sequence, or a sequence complementary therewith, when used to detect presence of other similar sequences in a sample. The detection is carried out by identification of 'hybridization complexes between the probe and the assayed sequence.
  • the probe may be attached to a solid support or to a detectable label.
  • the present invention is based on the finding of novel cell adhesion molecules containing an N-terminal which has Ig-like C2 type repeats, and a C-terminal which contains a neuropilin-like MAM domain.
  • the present invention provides by its first aspect, a novel isolated nucleic acid molecule comprising or consisting of the sequence of SEQ ID NO: 1 to SEQ ID NO: 7, fragments of said sequence having at least 20 nucleic acids, or a molecule comprising a sequence having at least 70%, preferably 80%, and most preferably 90% identity to SEQ ID NO:l to SEQ ID NO: 7.
  • the present invention further provides a protein or polypeptide comprising or consisting of an amino acid sequence encoded by any of the above nucleic acid sequences, termed herein "CAM-H product", for example, an amino acid sequence having the sequence as depicted in SEQ ID NO: 8 to 13, fragments of the above amino acid sequence having a length of at least 10 amino acids, as well as homologs of the amino acid sequences of SEQ ID NO: 8 to 13 in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified.
  • the present invention further provides nucleic acid molecule comprising or consisting of a sequence which encodes the above amino acid sequences, (including the fragments and analogs of the amino acid sequences).
  • nucleic acid sequences can code for the amino acid sequence of the invention.
  • Those alternative nucleic acid sequences which code for the same amino acid sequences codes by the sequences of SEQ ID NO: 1 to SEQ ID NO: 7 are also an aspect of the of the present invention.
  • the present invention further provides expression vectors and cloning vectors comprising any of the above nucleic acid sequences, as well as host cells trans fected by said vectors.
  • the present invention still further provides pharmaceutical compositions comprising, as an active ingredient, said nucleic acid molecules, said expression vectors, or said protein or polypeptide.
  • These pharmaceutical compositions are suitable for the treatment of diseases and pathological conditions, which can be ameliorated, cured or prevented by raising the level of the CAM-H product.
  • diseases and pathological conditions which can be ameliorated, cured or prevented by raising the level of the CAM-H product.
  • diseases which are manifested by non-normal levels of various CAMs, which are usually lower than normal levels of CAM, or alternatively, diseases in which the level of CAM is normal, but a therapeutically beneficial effect may be achieved by raising the level of CAM to a higher than normal level.
  • these diseases are concerned with interaction of cells at the extracellular matrix, or interaction with other cells which can lead to a plurality of diseases and pathological conditions, which are specified hereinbelow.
  • the pharmaceutical compositions of the invention may stimulate growth and regeneration of nerve cell axons in order to compensate for defects caused by genetic, inflammatory, infectious, neurogenerative or trauma causes, for example to target neurite regrowth to target muscles and nerve cells after nerve injury.
  • the composition may be used for the treatment of a plurality of CAM- involved diseases such as: inflammatory diseases, autoimmune diseases (Crohn's disease, colitis, rheumatoid arthritis), graft vs. host and host vs. graft diseases, for the treatment of multiple sclerosis, diabetes, atherosclerosis, various types of cancer, for the treatment of injuries caused by head trauma as well as for the treatment of various viral disease and the treatment of various respiratory diseases.
  • the present invention provides a nucleic acid molecule comprising or consisting of a non-coding sequence which is complementary to that of SEQ ID NO: 1 to SEQ ID NO: 7, or complementary to a sequence having at least 70% identity to said sequence or a fragment of said two sequences.
  • the complementary sequence may be a DNA sequence which hybridizes with SEQ ID NO: 1 to SEQ ID NO: 7, or hybridizes to a portion of that sequence having a length sufficient to inhibit the transcription of the complementary sequence.
  • the complementary sequence may be a DNA sequence which can be transcribed into an mRNA being an antisense to the mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO: 7 or into an mRNA which is an antisense to a fragment of the mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO: 7 which has a length sufficient to hybridize with the mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO: 7, so as to inhibit its translation.
  • the complementary sequence may also be the mRNA or the fragment of the mRNA itself.
  • the nucleic acid sequences of the second aspect of the invention may be used for therapeutic or diagnostic applications for example for detection of the expression of CAM-H in various tissues, such as neuronal tissue, endothelial or epithelial tissue, tissues connected to the immune system, tissue obtained from tumors as well as body fluids such as cerebrospinal fluid, plasma and blood.
  • Said detection may be indicative of a plurality of diseases, and pathological conditions stemming from genetic, inflammatory, infectious, degenerative or trauma causes.
  • the diseases which can be detected are the same diseases mentioned above in connection with the treatment by the therapeutical compositions of the invention above.
  • the detection may be indicative to the presence of tumors in general, and in particular to tumors from neuro-endocrine origin basis.
  • the level of any one of the CAM-H of the invention may be indicative of the invasiveness of the tumor indicating its stage of malignancy, which determination may be important for the prognosis of the patient, and for determining his/her best therapeutical modality.
  • the present invention also provides expression vectors comprising any one of the above defined complementary nucleic acid sequences and host cells transfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention.
  • the invention also provides anti-CAM-H product antibodies, namely antibodies directed against the CAM-H product which specifically bind to said CAM-H product as well as antibodies which can be obtained through cDNA vaccination.
  • Said antibodies are useful both for diagnostic and therapeutic purposes.
  • they may be indicative of neuronal diseases and pathological conditions (such as for example inflammatory and autoimmune diseases, diseases of the respiratory and vascular tract, and indicative of tumors of neuro-endocrine origin and for the stage of malignancy as indicated above.
  • said antibody may be as an active ingredient in a pharmaceutical composition as will be explained below.
  • the present invention also provides pharmaceutical compositions comprising, as an active ingredient, the nucleic acid molecules which comprise or consist of said complementary sequences, or of a vector comprising said complementary sequences.
  • the pharmaceutical composition thus provides pharmaceutical compositions comprising, as an active ingredient, said anti-CAM-H product antibodies.
  • compositions comprising said anti-CAM-H product antibodies or the nucleic acid molecule comprising said complementary sequence, are suitable for the treatment of diseases and pathological conditions where a therapeutically beneficial effect may be achieved by neutralizing the CAM-H or decreasing the amount of the CAM-H product or blocking its binding to its target (for example the ligand to which it binds on cells), for example, by the neutralizing effect of the antibodies, or by the decrease of the effect of the antisense mRNA in decreasing expression level of the CAM-H product.
  • diseases are manifested by a higher than normal level of the CAM-H of the invention, or by normal level of CAMs, however the disease may be ameliorated or a beneficial effect may be evident by decreasing said level.
  • diseases are for example tumors, and in particular of a neuro-endocrine origin, in which the invasiveness of the tumor is dependent on interactions between the CAM-H of the invention and basal membranes or extracellular matrixes, diseases involving the immune system as well as endothelial and epithelial membranes, as well as other diseases of neural origin involving cell-adhesion molecules.
  • the present invention provides methods for detecting the level of the transcript (mRNA) of said CAM-H product in a body fluid sample, plasma, cerebrospinal fluid, or in a specific tissue sample, for example by use of probes comprising or consisting of said coding sequences; as well as methods for detecting levels of expression of said product in tissue, e.g. by the use of antibodies capable of specifically reacting with the above amino acid sequences.
  • Detection of the level of the expression of the CAM-H of the invention may be indicative of a plurality of physiological or pathological conditions, as explained above.
  • the method for detection of a nucleic acid sequence which encodes the CAM-H product in a biological sample, comprises the steps of:
  • hybridization complexes wherein the presence of the complex indicates the presence of nucleic acid sequence encoding the CAM-H product in the biological sample.
  • the amount of hybridization complexes may be determined and calibrated by comparing it to a calibration scale in order to determine the amount of the nucleic acid sequence which enables the CAM-H product in the sample.
  • the level of each of the sequences SEQ ID NO: 1 to SEQ ID NO: 7 may be detected and either compared to each other, and said ratio may also be indicative to a plurality of pathological or physiological conditions as explained above.
  • the probe is part of a nucleic acid chip used for detection purposes, i.e. the probe is a part of an array of probes each present in a known location on a solid support.
  • the nucleic acid sequence used in the* above method may be a DNA sequence an RNA sequence, etc; it may be a coding or a sequence or a sequence complementary thereto (for respective detection of RNA transcripts or coding-DNA sequences).
  • Methods for detecting mutations in the region coding for the CAM-H product are also provided, which may be methods carried-out in a binary fashion,
  • CAM-H nucleic acid sequence and the one present in the sample, or carried-out by specifically detecting the nature and location of the mutation.
  • the present invention also concerns a method for detecting CAM-H product both for determining its presence, as well as its level or alterations in its level in a biological sample, comprising the steps of:
  • the present invention also concerns a method for detecting anti-CAM-H antibodies in a biological sample comprising the steps of: (a) contacting said biological sample with the product of the invention thereby forming an antibody-antigen complex; and
  • diseases are detected not by detecting the presence of the protein (product) which caused the disease, but rather by detecting the presence in a biological sample (such as blood or serum) of antibodies against such a product.
  • a biological sample such as blood or serum
  • the method of detecting the presence of anti-CAM-H antibodies is intended to be used in such case.
  • the amount of the antibody-antigen complex can be quantitized, in order to determine the level of the CAM-H-product or the anti-CAM-H antibodies, as the case may be.
  • the level of any of the products of SEQ ID NO: 8 may be compared to each level, and the ratio between the levels may be indicative to a plurality of physiological and pathological conditions as explained above.
  • the indicative ratio may not be the ratio of the proteins themselves but rather the ratio of antibodies against the proteins.
  • the ratio of the level of the mRNA transcripts of any one of SQ ID NO: 1 to SEQ ID NO: 7, and changes in said ratio may be indicative of a plurality of diseases or pathological conditions especially as detailed above.
  • the invention also provides a method for identifying candidate compounds capable of modulating the activity of CAM-H product (being either activators or deactivators).
  • the method includes: (i) providing a protein or polypeptide comprising an amino acid sequence substantially as depicted in SEQ ID NO: 8 to SEQ ID NO: 13, or a fragment of such a sequence;
  • the activity of the amino acid which should be changed by the modulator may be for example the binding of the CAM-H product to a ligand of an adhesion molecule present for example on the external cell surface. Any modulator which changes such an activity has a potential as serving as an actuator or deactivator.
  • the present invention also concerns compounds identified by the above methods described above, which compound may either be an activator of the CAM-H product or a deactivator thereof.
  • Fig. 1 is the alignment of the CAM-H product of SEQ ID NO: 3 (NCAM_c_l), 4 (NCAM_c_2); 5 (NCAM_d_2) and 6 (NCAM_d_l);
  • Fig. 2 is an alignment of human (SEQ ID NO: 4) and mouse (SEQ ID NO: 7) sequences;
  • Fig. 3 is an alignment of amino acid sequences of SEQ ID NO: 8 and 9;
  • Fig. 4 is an alignment of SEQ ID NOS: 10, 11, 12, 13;
  • Fig. 5 shows gel of PCR fragment of the sequence of the invention in various tissues; and
  • Fig. 6 shows a schematic drawing of the amino acid sequence of SEQ ID NO: 8 and 9;
  • the nucleic acid sequences of the invention include nucleic acid sequences which encode CAM-H product and fragments and analogs thereof.
  • the nucleic acid sequences may alternatively be sequences complementary to the above coding sequence, or to a region of said coding sequence. The length of the complementary sequence is sufficient to avoid the expression of the coding sequence.
  • the nucleic acid sequences may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA.
  • the DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.
  • the nucleic acid sequences may also both include dNTPs, rNTPs as well as non naturally occurring sequences.
  • the sequence may also be a part of a hybrid between an amino acid sequence and a nucleic acid sequence.
  • the nucleic acid sequence has at least 70%, preferably 80% or 90% sequence identity with.the sequences identified as SEQ ID NO: 1 to SEQ ID NO: 7.
  • the nucleic acid sequences may include the coding sequence by itself.
  • the coding region may be in combination with additional coding sequences, such as those coding for fusion protein or signal peptides, in combination with non-coding sequences, such as introns and control elements, promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or in a vector or host environment in which the CAM-H nucleic acid sequence is introduced as a heterologous sequence.
  • the nucleic acid sequences of the present invention may also have the product coding sequence fused in-frame to a marker sequence which allows for purification of the CAM-H product.
  • the marker sequence may be, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. Cell 37:767 (1984)).
  • fragments also referred to herein as oligonucleotides, typically having at least 20 bases, preferably 20-30 bases corresponding to a region of the coding-sequence nucleic acid sequence.
  • the fragments may be used as probes, primers, and when complementary also as antisense agents, and the like, according to known methods.
  • the nucleic acid sequence may be substantially a depicted in SEQ ID NO: 1 to SEQ ID NO: 7 or fragments thereof or sequences having at least 70%, preferably 70-80%), most preferably 90% identity to the above sequence.
  • the sequence may be a sequence coding the amino acid sequence of SEQ ID NO: 8 to SEQ ID NO: 13, or fragments or analogs of said amino acid sequence.
  • the nucleic acid sequences may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify nucleic acid sequences which encode the CAM-H products disclosed above.
  • cDNA libraries prepared from a variety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
  • the nucleic acid sequences may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3' untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerous methods known to those of skill in the art, such as PCR or primer extension (Sambrook et al, supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # Kl 802-1).
  • genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al, Nucleic Acids Res. 16:8186, (1988)).
  • the primers may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992; National Biosciences Inc, Madison, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Capture PCR (Lagerstrom, M. et al, PCR Methods Applic 1: 111-19, (1991)) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR.
  • flanking sequences Another method which may be used to retrieve flanking sequences is that of Parker, J.D., et al, Nucleic Acids Res., 19:3055-60, (1991)). Additionally, one can use PCR, nested primers and PromoterFinderTM libraries to "walk in" genomic DNA (PromoterFinderTM; Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions. Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
  • a randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
  • the nucleic acid sequences and oligonucleotides of the invention can also be prepared by solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to form continuous sequences up to several hundred bases.
  • nucleic acid sequences specified above may be used as recombinant DNA molecules that direct the expression of CAM-H products.
  • Codons preferred by a particular prokaryotic or eukaryotic-Host can be selected, for example, to increase the rate of CAM-H product expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • nucleic acid sequences of the present invention can be engineered in order to alter a CAM-H product coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the product.
  • alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, et al, (supra).
  • the present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of the product of the invention by recombinant techniques.
  • Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the expression of the CAM-H nucleic acid sequence.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
  • the nucleic acid sequences of the present invention may be included in any one of a variety of expression vectors for expressing a product.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such promoters include: LTR or SV40 promoter, the E.coli lac or trp promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed Jiost cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E.coli.
  • the vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate expression hosts include: bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses; plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. The invention is not limited by the host cells employed.
  • a number of expression vectors may be selected depending upon the use intended for the CAM-H product. For example, when large quantities of CAM-H product are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, multifunctional E.coli cloning and expression vectors such as Bluescript(R) (Stratagene), in which the CAM-H polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster J. Biol. Chem. 264:5503-5509, (1989)); pET vectors (Novagen, Madison WI); and the like.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • the expression of a sequence encoding CAM-H product may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al, Nature 310:511-514. (1984)) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al, EMBO J., 3:17-311, (1987)).
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al., EMBO J.
  • CAM-H product may also be expressed in an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the CAM-H product coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of CAM-H coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
  • a number of viral-based expression systems may be utilized.
  • a CAM-H product coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing CAM-H protein in infected host cells (Logan and Shenk, Proc. Natl. Acad. Sci. 81:3655-59, (1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • Specific initiation signals may also be required for efficient translation of a CAM-H protein coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where CAM-H product coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D. et al, (1994) Results Probl. Cell Differ., 20: 125-62, (1994); Bittner et al., Methods in Enzymol 153:516-544, (1987)).
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology).
  • Cell-free translation systems can also be employed to produce polypeptides using RNAs derived from the DNA constructs of the present invention.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which, cleaves a "pre-pro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express CAM-H product may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M., et al, Cell 11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I., et al., Cell 22:817-23, (1980)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M., et al, Proc. Natl. Acad. Sci.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al, J. Mol Biol, 150: 1-14, (1981)) and als ox pat, which confer resistance to CAM-Horsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman S.C. and R.C. Mulligan, Proc. Natl.
  • Host cells transformed with a nucleotide sequence encoding CAM-H product may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the product produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing nucleic acid sequences encoding CAM-H product can be designed with signal sequences which direct secretion of CAM-H product through a prokaryotic or eukaryotic cell membrane.
  • CAM-H product may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.).
  • the inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and CAM-H protein is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein compromising a CAM-H polypeptide fused to a polyhistidine region separated by an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath, et al., Protein Expression and Purification, 3:263-281, (1992)) while the enterokinase cleavage site provides a means for isolating CAM-H polypeptide from the fusion protein.
  • pGEX vectors Promega, Madison, Wis.
  • GST S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
  • ligand-agarose beads e.g., glutathione-agarose in the case of GST-fusions
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
  • the CAM-H products can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • nucleic acid sequences of the present invention may be used for a variety of diagnostic purposes.
  • the nucleic acid sequences may be used to detect and quantitate expression of CAM-H in patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for CAM-H product.
  • the assay may be used to detect soluble CAM-H in the serum or blood. This assay typically involves obtaining total mRNA from the tissue or serum and contacting the mRNA with a nucleic acid probe.
  • the probe is a nucleic acid molecule of at least 20 nucleotides, preferably 20-30 nucleotides, capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding CAM-H under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of CAM-H.
  • This assay can be used to distinguish between absence, presence, and excess expression of CAM-H product and to monitor levels of CAM-H expression during therapeutic intervention.
  • the invention also contemplates the use of the nucleic acid sequences as a diagnostic for diseases resulting from inherited defective CAM-H sequences. These sequences can be detected by comparing the sequences of the defective (i.e., mutant) CAM-H coding region with that of a normal coding region. Association of the sequence coding for mutant CAM-H product with abnormal CAM-H product activity may be verified. In addition, sequences encoding mutant CAM-H products can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, complementation experiments in a CAM-H protein deficient strain of HEK293 cells) as yet another means to verify or identify mutations.
  • a functional assay system e.g., colorimetric assay, complementation experiments in a CAM-H protein deficient strain of HEK293 cells
  • nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al., Nature 324: 163-166, (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a, change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et alProc. Natl. Acad. Sci. USA, 85:4397-4401, (1985)), or by differences in melting temperatures. "Molecular beacons" (Kostrikis L.G.
  • hairpin-shaped, single-stranded synthetic oligo- nucleotides containing probe sequences which are complementary to the nucleic acid of the present invention may also be used to detect point mutations or other sequence changes as well as monitor expression levels of CAM-H product. Such diagnostics would be particularly useful for prenatal testing.
  • Another method for detecting mutations uses two DNA probes which are
  • __* designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutation(s) is at or near the abutting bases.
  • the two probes may be joined at the abutting bases, e.g., in the presence of a ligase enzyme, but only if both probes are correctly base paired in the region of probe junction.
  • the presence or absence of mutations is then detectable by the presence or absence of ligated probe.
  • oligonucleotide array methods based on sequencing by hybridization (SBH), as described, for example, in U.S. Patent No. 5,547,839.
  • SBH sequencing by hybridization
  • the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip.
  • the sequence of the target can then be "read" from the pattern of target binding to the array.
  • the nucleic acid sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 20-30 bp) from the CAM-H cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids or using instead radiation hybrids are rapid procedures for assigning a particular DNA to a particular chromosome.
  • mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 50 or 60 bases.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map data are found, for example, in the OMIM database (Center for Medical Genetics, Johns Hopkins University, Baltimore, MD and National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD).
  • the OMIM gene map presents the cytogenetic map location of disease genes and other expressed genes.
  • the OMIM database provides information on diseases associated with the chromosomal location. Such associations include the results of linkage analysis mapped to this interval, and the correlation of translocations and other chromosomal aberrations in this area with the advent of various diseases associated with abnormal amounts or function of various CAM proteins.
  • Nucleic acid sequences of the invention may also be used for therapeutic purposes.
  • expression of CAM-H product may be modulated through antisense technology, which controls gene expression through hybridization of complementary nucleic acid sequences, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding CAM-H product.
  • the 5' coding portion of the nucleic acid sequence sequence which codes for the product of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs in length.
  • Oligonucleotides derived from the transcription CAM-Ht site e.g.
  • An antisense DNA oligonucleotide is designed to be complementary to a region of the nucleic acid sequence involved in transcription (Lee et al, Nucl. Acids, Res., 6:3073, (1979); Cooney et al., Science 241:456, (1988); and Dervan et al, Science 251: 1360, (1991)), thereby preventing transcription and the production of the CAM-H products.
  • An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the CAM-H products (Okano J. Neurochem. 56:560, (1991)).
  • the antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may be expressed in vivo.
  • the antisense may be antisense mRNA or DNA sequence capable of coding such antisense mRNA.
  • the antisense mRNA or the DNA coding thereof can be complementary to the full sequence of nucleic acid sequences coding to the CAM-H protein or to a fragment of such a sequence which is sufficient to inhibit production of a protein product.
  • expression of CAM-H product may be increased by providing coding sequences for coding for said product under the control of suitable control elements ending its expression in the desired host.
  • the nucleic acid sequences of the invention may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • Cells from a patient may be engineered with a nucleic acid sequence (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA nucleic acid sequence
  • Such methods are well-known in the art.
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retro viruses from which the retroviral plasmid vectors mentioned above may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller (Human Gene Therapy, Vol. 1, pg. 5-14, (1990)).
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • the genes introduced into cells may be placed under the control of inducible promoters, such as the radiation-inducible Egr-1 promoter, (Maceri,
  • the substantially purified CAM-H product of the invention has been defined above as the product coded from the nucleic acid sequence of the invention.
  • the amino acid sequence is an amino acid sequence having at least 70%, preferably at least 80% or 90% identity to the sequence identified as SEQ ID NO: 8 to SEQ ID NO: 13.
  • the protein or polypeptide may be in mature and/or modified form, also as defined above. Also contemplated are protein fragments having at least 10 contiguous amino acid residues, preferably at least 10-20 residues, derived from the CAM-H product.
  • sequence variations are preferably those that are considered conserved substitutions, as defined above.
  • a protein with a sequence having at least 80%> sequence identity with the protein identified in SEQ ID NO: 8 to SEQ ID NO: 13, preferably by utilizing conserved substitutions as defined above is also part of the invention.
  • the protein has or contains the sequence identified as SEQ ID NO: 8 to SEQ ID NO: 13.
  • the CAM-H product may be (i) one in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the CAM-H product is fused with another compound, such as a compound to increase the half- life of the protein (for example, polyethylene glycol (PEG)), or a moiety which serves as targeting means to direct the protein to its target tissue or target cell population (such as an antibody), or (iv) one in which additional amino acids are fused to the CAM-H product.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • another compound such as a compound to increase the half- life of the protein (for example, polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • fragments and portions of CAM-H product may be produced by direct peptide synthesis using solid-phase techniques (cf. Stewart et al, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J., J. Am. Chem. Soc, 85:2149-2154, (1963)).
  • In vitro peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems
  • CAM-H expression and or diseases which can be cured or ameliorated by raising the level of the CAM-H product, even if the level is normal.
  • diseases where non-normal growth, regeneration or tumors of neurons is evidenced due to genetic, degenerative or injury causes.
  • these diseases are in CAM-H products or fragments and may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue.
  • the product-containing compositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones.
  • CAM-H product-containing compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means as well as by nasal application.
  • CAM-H product-containing compositions may also be administered via liposomes.
  • Such administration routes and appropriate formulations are generally known to those of skill in the art.
  • the product can be given via intravenous or intraperitoneal injection.
  • the product may be injected to other localized regions of the body.
  • the product may also be administered via nasal insufflation. Enteral administration is also possible.
  • the product should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
  • a therapeutic composition for use in the treatment method can include the product in a sterile injectable solution, the polypeptide in an oral delivery vehicle, the product in an aerosol suitable for nasal administration, or the product in a nebulized form, all prepared according to well known methods.
  • Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the product of the invention may also be used to modulate endothelial differentiation and proliferation as well as to modulate apoptosis either ex vivo or in vitro, for example, in cell cultures.
  • Example III Screening methods for activators and deactivators (inhibitors)
  • the present invention also includes an assay for identifying molecules, such as synthetic drugs, antibodies, peptides, dr other molecules, which have a modulating effect on the activity of the CAM-H product, e.g. activators or deactivators of the CAM-H product of the present invention.
  • an assay comprises the steps of providing an CAM-H product encoded by the nucleic acid sequences of the present invention and determining its physiological activity on the target in the presence and absence of one or more candidate molecules to determine the candidate molecules. Those molecules which are modulating effect on the activity of the CAM-H product are selected as likely candidates for activators and deactivators.
  • CAM-H product its catalytic or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell membrane or located intracellularly.
  • the formation of binding complexes, between CAM-H product a d ⁇ the agent being tested, may be measured.
  • the activator or deactivator may work by serving as agonist or antagonist, respectively, of the CAM-H receptor and their effect may be determined in connection with the receptor.
  • CAM-H product Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the CAM-H product is described in detail by Geysen in PCT Application WO 84/03564, published on Sep. 13, 1984.
  • large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with the full CAM-H product or with fragments of CAM-H product and washed. Bound CAM-H product is then detected by methods well known in the art.
  • Substantially purified CAM-H product can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • Antibodies to the CAM-H product as described in Example IV below, may also be used in screening assays according to methods well known in the art.
  • a "sandwich" assay may be performed, in which an anti-CAM-H antibody is affixed to a solid surface such as a microtiter plate and CAM-H product is added.
  • a solid surface such as a microtiter plate
  • CAM-H product is added.
  • Such an assay can be used to capture compounds which bind to the CAM-H product.
  • such an assay may be used to measure the ability of compounds to influence with the binding of CAM-H product to the CAM-H receptor and then select those compounds which effect the binding.
  • the purified CAM-H product is used to produce anti-CAM-H antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of the CAM-H product, in particular therapeutic applications in modulating the effect of CAM-H on moieties to which it binds in the extracellular matrix.
  • Antibodies to CAM-H product may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which inhibit dimer formation, are especially preferred for therapeutic use.
  • a fragment CAM-H product for antibody induction does not require biological activity but have to feature immunological activity; however, the protein fragment or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids of the sequences specified in SEQ ID NO: 8 to SEQ ID NO: 13.
  • CAM-H protein amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced ⁇ against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to CAM-H product.
  • various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with CAM-H product or any portion, fragment or oligopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to CAM-H protein may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497, (1975)), the human B-cell hybridoma technique (Kosbor et al, Immunol. Today 4:72, (1983); Cote et al, Proc Natl. Acad. Sci. 80:2026-2030, (1983)) and the EBV-hybridoma technique (Cole, et al, Mol. Cell Biol. 62: 109-120, (1984)).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (Proc Natl. Acad. Sci. 86:3833-3837, 1989)), and Winter G and Milstein C, (Nature 349:293-299, (1991)).
  • Antibody fragments which contain specific binding sites for CAM-H protein may also be generated.
  • fragments include, but are not limited to, the F(ab') fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al, Science 256: 1275-1281, (1989)).
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific CAM-H product is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D.E., et al, (J Exp. Med. 158: 1211, (1983)).
  • Antibodies which specifically bind CAM-H product are useful for the diagnosis of conditions or diseases characterized by over or under expression of CAM-H. Alternatively, such antibodies may be used in assays to monitor patients being treated with CAM-H product, its activators, or its deactivators.
  • Diagnostic assays for CAM-H protein include methods utilizing the antibody and a label to detect CAM-H product in human body fluids or extracts of cells or tissues.
  • the products and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule.
  • reporter molecules A wide variety of reporter molecules are known in the art.
  • a variety of protocols for measuring CAM-H product, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on CAM-H product is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al. (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of CAM-H product expression.
  • Normal or standard values for CAM-H product expression are established by combining body or cell extracts taken from normal subjects, preferably human, with antibody to CAM-H product under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by various methods, preferably by photometric methods.
  • standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
  • the antibody assays are useful to determine the level of CAM-H present in a body fluid sample, in order to determine whether it is being overexpressed or underexpressed in the tissue, or as an indication of how CAM-H levels are responding to drug treatment.
  • Another alternative is to determine the presence and/or level of naturally occurring anti-CAM-H antibodies in a sample, such as blood or serum. Many times diseases are identified by detecting the presence or level of antibodies against a specific product. For the detection of such naturally occurring anti-CAM-H antibodies, the sample may be contacted with the product of the invention, for example as depicted in SEQ ID NO: 3 or SEQ ID NO: 4, or with an antigenic fragment thereof, and the presence or level of antibody-antigen complexes may be determined by methods well known in the art.
  • the antibodies may have a therapeutical utility in blocking or decreasing the activity of the CAM-H product in pathological conditions where beneficial effect can be achieved by such a decrease.
  • the antibody employed is preferably a humanized monoclonal antibody, or a human Mab produced by known globulin-gene library methods.
  • the antibody is administered typically as a sterile solution by IV injection, although other parenteral routes may be suitable.
  • the antibody is administered in an amount between about 1-15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1-7 days, until a therapeutic improvement is seen.
  • RNA Five ⁇ g of total RNA were isolated from human tissues. The reaction was performed in a final volume of 20 ⁇ l and also contained Superscript II Reverse Transcriptase (Gibco/BRL, Gaithersburg, MD), lxBuffer supplied by the manufacturer, 30 units of Rnasin (Promega, Medison, WI) and 10 pmol of oligoDT (Promega, Medison, WI).
  • Superscript II Reverse Transcriptase Gibco/BRL, Gaithersburg, MD
  • lxBuffer supplied by the manufacturer
  • 30 units of Rnasin Promega, Medison, WI
  • 10 pmol of oligoDT Promega, Medison, WI.
  • the cycling conditions were 94°C-3 min. Taq polymerase followed by 30 cycles of 94°C for 30 sec, 68°C-1 min and final extension at 68°C for 10 mins.
  • the PCR reaction on PTC-225 (MJ Research, Inc).
  • Fig. 5 The resulting sequences were separated on a gel and the results are shown in Fig. 5. As can be seen the transcript (from SEQ ID NO: 3) was identified in Brain Cerebellum and was not identified in tissues obtained from other tissues proving that the sequences of the invention are indeed unique to brain.

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EP00969784A 1999-10-19 2000-10-19 N-cam homologe und dessen spleissvarianten Withdrawn EP1230358A2 (de)

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IL13245999 1999-10-19
IL13245999A IL132459A0 (en) 1999-10-19 1999-10-19 Brain specific sequences
IL13493200 2000-03-07
IL13493200A IL134932A0 (en) 2000-03-07 2000-03-07 Novel nucleic acid and amino acid sequences
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