EP1159431A2 - Human kallikrein-like genes - Google Patents

Human kallikrein-like genes

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Publication number
EP1159431A2
EP1159431A2 EP00908878A EP00908878A EP1159431A2 EP 1159431 A2 EP1159431 A2 EP 1159431A2 EP 00908878 A EP00908878 A EP 00908878A EP 00908878 A EP00908878 A EP 00908878A EP 1159431 A2 EP1159431 A2 EP 1159431A2
Authority
EP
European Patent Office
Prior art keywords
klk
protein
gene
nucleic acid
genes
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.)
Withdrawn
Application number
EP00908878A
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German (de)
English (en)
French (fr)
Inventor
George M. Yousef
Eleftherios P. Diamandis
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Mt Sinai Hospital
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Mt Sinai Hospital
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Publication date
Application filed by Mt Sinai Hospital filed Critical Mt Sinai Hospital
Priority to EP06076156A priority Critical patent/EP1724351A3/en
Publication of EP1159431A2 publication Critical patent/EP1159431A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to nucleic acid molecules, proteins encoded by such nucleic acid molecules, and use of the proteins and nucleic acid molecules BACKGROUND OF THE INVENTION
  • Kailikreins and kallikrein- ke proteins are a subgroup of the serine protease enzyme family and exhibit a high degree of substrate specificity (1)
  • the biological role of these kailikreins is the selective cleavage of specific polypeptide precursors (substrates) to release peptides with potent biological activity (2)
  • kailikreins are encoded by large multigene families In the mouse genome, at least 24 genes have been identified (3) Expression of 11 of these genes has been confirmed, the rest are presumed to be pseudogenes (4)
  • a similar family of 15-20 kailikreins has been found in the rat genome (5) where at least 4 of these are known to be expressed (6)
  • PSA prostatic specific antigen
  • KLK2 human glandular kallikrem
  • KLKl tissue (pancreatic-renal) kalhkrem
  • the mouse kal krem genes are clustered in groups of up to 11 genes on chromosome 7 and the distance between the genes in the various clusters can be as small as 3-7 Kb (3) All three human kalhkrein genes have been assigned to chromosome 19q 13 2 - 19q 13 4 and the distance between PSA and KLK2 has been estimated to be 12 Kb (9)
  • KLK-L3, KLK-L4, KLK-L5, or KLK-L6 and collectively as “kallikrein-hke proteins” oi "KLK-L Proteins”
  • the genes encoding the proteins are referred to as " Uk-ll klk-12 lclk-13 klk-14 klk-1 oi klk 16 ", and collectively as “kallikrein-hke genes” or "klk-I genes"
  • the present invention relates to an isolated nucleic acid molecule which comprises (l) a nucleic acid sequence encoding a protein having substantial sequence identity with an amino acid sequence of KLK-L 1, KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 as shown m SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, (u) a nucleic acid sequence encoding a protein comprising an ammo acid sequence of KLK-
  • KLK-L2, KLK-L3, KLK-L4, KLK-L5, or KLK-L6 as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively,
  • a nucleic acid sequence capable of hybridizing under stringent conditions to a nucleic acid sequence in (I), (n) or (in), (vi) a nucleic acid sequence encoding a truncation, an analog, an allelic or species variation of a protein comprising an amino acid sequence of KLK-L1, KLK-L2, KLK-L3, KLK- L4, KLK-L5, or KLK-L6 as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, respectively, or (vn) a fragment, or allelic or species variation of (l), (n) or (in)
  • a purified and isolated nucleic acid molecule of the invention comprises
  • nucleic acid sequence comprising the sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65 wherein T can also be U
  • nucleic acid sequences complementary to (l) preferably complementary to the full nucleic acid sequence of SEQ ID NO 1, 13, 21, 43, 56, or 65
  • a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (I) or (n) and preferably having at least 18 nucleotides
  • a nucleic acid molecule differing from any of the nucleic acids of (I) to (in) in codon sequences due to the degeneracy of the genetic code
  • the invention also contemplates a nucleic acid molecule comprising a sequence encoding a truncation of a KLK-L protein, an analog, or a homolog of a KLK-L Protein or a truncation thereof (KLK-
  • KLK-L Related Proteins L Proteins and truncations, analogs and homologs of KLK-L Proteins are also collectively referred to herein as "KLK-L Related Proteins"
  • nucleic acid molecules of the invention may be inserted into an appropriate expression vector, l e a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence
  • recombinant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements linked to the nucleic acid molecule
  • the recombinant expression vector can be used to prepare transformed host cells expressing KLK- L Related Proteins Therefore, the invention further provides host cells containing a recombinant molecule of the invention
  • the invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule comprising a nucleic acid molecule of the invention, in particular one which encodes an analog of a KLK-L Protein, or a truncation of a KLK-L Protein
  • the invention further provides a method for preparing KLK-L Related Proteins utilizing the purified and isolated nucleic acid molecules of the invention
  • a method for preparing a KLK-L Related Protein comprising (a) transferring a recombinant expression vector of the invention into a host cell, (b) selecting transformed host cells from untransformed host cells, (c) culturing a selected transformed host cell under conditions which allow expression of the KLK-L Related Protein, and (d) isolating the KLK-
  • the invention further broadly contemplates an isolated KLK-L Protein comprising an amino acid sequence as shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • the KLK-L Related Proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins This may be accomplished, for example, by the synthesis of N-terrrunal or C-terminal fusion proteins
  • the invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and/or to proteins of the invention Therefore, the invention also relates to a probe comprising a nucleic acid sequence of the invention, or a nucleic acid sequence encoding a protein of the invention, or a part thereof
  • the probe may be labeled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleic acid molecule of the invention including nucleic acid molecules coding for a protein which displays one or more of the properties of a protein of the invention
  • the invention still further provides a method for identifying a substance which binds to a protein of the invention comprising reacting the protein with at least one substance which potentially can bind with the protein, under conditions which permit the formation of complexes between the substance and protein and detecting binding Binding may be detected by assaying for complexes, for free substance, or for non- complexed protein
  • the invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with a KLK-L Related Protein Methods can also be utilized which identify compounds which bind to KLK-L gene regulatory sequences (e g promoter sequences)
  • the invention provides a method for evaluating a compound for its ability to modulate the biological activity of a KLK-L Related Protein of the invention
  • a substance which inhibits or enhances the interaction of the protein and a substance which binds to the protein may be evaluated
  • the method comprises providing a known concentration of a KLK-L Related Protein, with a substance which binds to the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes
  • Compounds which modulate the biological activity ot a protein ot the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of the protein of the invention in tissues and cells, in the presence, and in the absence of the compounds
  • proteins of the invention and substances and compounds identified using the methods of the invention, and peptides of the invention may be used to modulate the biological activity of a KLK-L
  • the substances and compounds may be formulated into compositions for administration to individuals suffering from cancer
  • the present invention also relates to a composition
  • a composition comprising one or more of a protein of the invention, a peptide of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, exc ⁇ ient or diluent
  • a method for treating or preventing cancer comprising administering to a patient m need thereof, a KLK-L Related
  • Protein of the invention or a composition of the invention
  • Figure 1 shows an approximate 300 Kb of contiguous genomic sequence around chromosome 19ql3 3 - ql3 4 represented by 8 contigs, each one shown with its length in Kb
  • the contig numbers refer to those reported in the Lawrence Livermore National Laboratory website Note the localization of the seven known genes (PSA, KLK2, Zyme, NES1, HSCCE, neuropsin and TLSP) (see abbreviations for full names of these genes) All genes are represented with arrows denoting the direction of transcription
  • the gene with no homology to human kailikreins is termed UG (unknown gene)
  • the five new kallikrein-hke genes (KLK-L 1 to KLK-L5) were numbered from the most centrome ⁇ c to the most telome ⁇ c Numbers just below or just above the arrows indicate appropriate Kb lengths in each contig Gene lengths and distances between genes are rounded to the nearest 6 5 kb
  • Figure 2 shows a contiguous
  • FIG. 3 shows tissue expression of the prostase/KLK-Ll gene as determined by RT-PCR Actin and PSA are control genes Interpretations are presented in Table 9
  • Figure 4 shows the sequence of PCR product obtained with cDNA from female breast tissue using prostase/KLK-Ll primers Primer sequences are underlined The sequence is identical to the sequence obtained from prostatic tissue
  • Figure 6 is a schematic diagram showing comparison of the genomic structure of PSA, KLKl,
  • KLK2, zyme, neuropsin and prostase/KLK-Ll genes Exons are shown by open boxes and introns by the connecting lines Arrow head shows the start codons and the vertical arrow represents stop codons Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases
  • the intron phase refers to the location of the intron within the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs
  • Figure 7 shows the genomic organization and partial genomic sequence of the KLK-L2 gene Intronic sequences are not shown except for the splice junctions. Introns are shown with lower case letters and exons with capital letters. The start and stop codons are encircled and the exon -intron junctions are boxed The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are inside triangles Putative polyadenylation signal is underlined
  • Figure 8 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19ql3.3- ql3.4. Genes are represented by horizontal arrows denoting the direction of the coding sequence Distances between genes are mentioned in base pairs
  • Figure 9 shows the alignment of the deduced amino acid sequence of KLK-L2 with members of the kal krein multi-gene family.
  • Genes are (from top to bottom) • Prostase/KLK-Ll, enamel matrix serine proteinase 1 (EMSPl) (GenBank accession # NP_004908), KLK-L2, zyme (GenBank accession # Q92876), neuropsin (GenBank accession # BAA28673), trypsin-hke serine protease (TLSP) (GenBank accession # B AA33404), PSA (GenBank accession # P07288), KLK2 (GenBank accession # P20151),
  • KLKl GenBank accession # NP_0022478
  • trypsinogen GenBank accession # P07477)
  • Dashes represent gaps to bring the sequences to better alignment
  • the residues of the catalytic triad are represented by (•---•) and the 29 invariant serine protease residues by (I or •---•)
  • conserveed areas around the catalytic triad are boxed
  • the predicted cleavage sites are indicated by ( )
  • the dotted area represents the kalhkrein loop sequence
  • the trypsin like cleavage pattern is indicated by ( ⁇ )
  • Figure 10(A) shows a dendrogram of the predicted phylogenetic tree for some kal krein genes
  • KLKl, KLK2, and PSA together and aligned the KLK-L2 gene in one group with EMSP, prostase, and TLSP (B) Plot of hydrophobicity and hydrophilicity of KLK-L2
  • Figure 11 is a blot showing tissue expression of KLK-L2 gene as determined by RT-PCR Actin and PSA are control genes Interpretations are presented in Table 12
  • Figure 13 are blots of EtBr-stained agarose gels Total RNA was extracted from normal, benign, and cancer tissues and used to generate cDNA PCR was performed on cDNA
  • Figure 14 shows an approximate 300 Kb region of almost contiguous genomic sequence around chromosome 19q I3 3- ql3 4 Genes are represented by horizontal arrows denoting the direction of the coding sequence Gene lengths and distances between genes are rounded to the nearest 05 kb The site of the gap is marked with an asterisk Telome ⁇ c to TLSP there are likely another three kallikrein-hke genes
  • Figure 15 shows the genomic organization and partial genomic sequence of the KLK-L3 gene
  • Intronic sequences are not shown except for the splice junctions Introns are shown with lower case letters and exons with capital letters For the full sequence, see SEQ ID NO 21 The start and stop codons are encircled and the exon -intron junctions are boxed The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are inside triangles Putative polyadenylation signal is underlined
  • Figure 16 is a plot of hydrophobicity and hydrophilicity, comparing the pattern of the KLK-L3 with that of the zyme gene Note the hydrophobic region around the first twenty amino acids, likely representing the signal peptide
  • Figure 17 is an alignment of the deduced amino acid sequence of KLK-L3 with members of the kalhkrein multi-gene family Genes are (from top to bottom and in brackets is the GenBank accession #) PSA (P07288), KLK2 (P20151), KLKl (NP002248), trypsinogen (P07477), KLK-L3 (AF135026), trypsin- like serine protease (TLSP) (BAA33404), neuropsin (BAA28673), zyme (Q92876), human stratum corneum chymotryptic enzyme (HSCCE) (AAD49718), and prostase/KLK-Ll (AAD21581) (See SEQ ID NOs 78 to 84) Dashes represent gaps to bring the sequences to better alignment The residues of the catalytic triad are bold and in italics, and the 29 invariant serine protease residues are denoted by ( ⁇
  • Cysteine residues are marked by (•) conserveed areas around the catalytic triad are highlited in black The arrow heads ( "* ⁇ ) represent the potential cleavage sites The dotted area represents the kalhkrein loop sequence
  • Figure 18 is a dendrogram of the predicted phylogenetic tree for some serine proteases and kal krein genes Neighbor-joimng/UPGMA method was used to align KLK-L3 with other members of the kalhkrein gene family Gene names and accession numbers are listed in Figure 17 The tree grouped the classical kailikreins (KLKl , KLK2, and PSA) together and aligned the KLK-L3 gene in one group with TLSP, neuropsin, and NES 1 genes KLK-L4 (SEQ ID NO 43) lies further telome ⁇ c to TLSP (21)
  • Figure 19 is a blot showing tissue expression of the KLK-L3 gene as determined by RT-PCR Actin and PSA are control genes
  • Figure 21 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES1, and KLK-L3 genes Exons are shown by black boxes and introns by the connecting lines Arrowheads show the start codon, and arrows show the stop codon Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases
  • the intron phase refers to the location of the intron within the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons
  • Numbers inside boxes indicate exon lengths base pairs
  • Figure 22 shows a comparative genomic structure of the ESTs (Table 16), the clone from The German Genome Project, and the long form of KLK-L4 Exons are represented by solid bars and introns by the connecting lines Exon numbers on top of solid bars
  • Figure 24 in the Upper Panel is a Diagram showing the comparative genomic structure of the long KLK-L4 form and the short KLK-L4 variant Exons are represented by boxes and introns by the connecting lines Exon numbers refer to SEQ ID NO 43 and GenBank Accession No AF135024
  • the black region indicates the extra fragment (214 bp) that is found in the long, but not in the short form of the gene
  • the positions of the stop codons of the two forms are marked with asterisks Frame shifting occurs as a result of utilization of an alternative splice site, and a stop codon is generated at the beginning of exon 4 in the short form
  • the Lower Panel shows PCR products of the amplification of the KLK-L4 gene using L4-R1 and L4-X1 primers ( Figure 22 and Table 15) Note the predominant long form and a minor band representing the short form of KLK-L4 mRNA (M), Markers with sizes in bp shown on the left Tissues used (1), salivary gland, (2), mamm
  • Figure 25 shows the genomic organization and partial genomic sequence of the KLK-L4 gene Intronic sequences are not shown except for the splice junction areas Introns are shown with lower case letters and exons with capital letters For full sequence, see SEQ ID NO 43 or GenBank Accession
  • Figure 26 is a plot of hydrophobicity and hydrophilicity of the KLK-L4 protein, as compared with the glandular kal krein gene 2 (KLK2) Note the hydrophobic region at the amino terminus, suggesting presence of a signal peptide
  • Figure 27 shows an alignment of the deduced am o acid sequence of KLK-L4 with members of the kalhkrein multi-gene family Genes are (from top to bottom, and in brackets are the GenBank accession #) KLK-L 1/prostdse (AAD21581).
  • Figure 31 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES 1, and KLK-L4 genes Exons are shown by black boxes and introns by the connecting lines
  • the arrowhead shows the start codons and the arrow the stop codons
  • Letters above boxes indicate the relative positions of the amino acids of the catalytic triad
  • H denotes histidine
  • D aspartic acid and S serine Roman numbers indicate intron phases
  • the intron phase refers to the location of the intron within the codon, I, the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons
  • Numbers inside boxes indicate exon lengths in base pairs The question mark indicates the possibility of more untranslated bases
  • Figure 32 is a diagram showing the comparative genomic structure of the three splice forms of KLK-L5, the classical kalhkrein form, related protein- 1, and related prote ⁇ n-2 Exons are represented by solid bars and introns by the connecting lines Exon numbers refer to SEQ ID NO 56 and GenBank
  • Figure 33 shows the genomic organization and partial genomic sequence of the KLK-L5 gene
  • Intronic sequences are not shown except for short sequences around the splice junctions Introns are shown with lower case letters and exons with capital letters For full sequence, see SEQ ID NO 56 The start and stop codons are encircled and the exon -intron junctions are underlined The translated amino acids of the coding region are shown underneath by a single letter abbreviation The catalytic residues are boxed
  • Figure 34 is a schematic diagram showing the comparison of the genomic structure of PSA, KLK2, neuropsin, NES1, KLK-L4 and KLK-L5 genes Exons are shown by solid bars and introns by the connecting lines Arrowhead marks the site of the start codon, and the arrow represents the stop codon Letters above boxes indicate relative positions of the catalytic triad, H denotes histidine, D aspartic acid and S serine Roman numbers indicate intron phases The intron phase refers to the location of the intron with the codon, I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, 0 the intron occurs between codons Numbers inside boxes indicate exon lengths in base pairs Question marks indicate that exact length is not accurately known
  • Figure 35 is a plot of hydrophobicity and hydrophilicity of KLK-L5 protein compared to prostate specific antigen (PSA)
  • PSA prostate specific antigen
  • Figure 36 shows an alignment of the deduced ammo acid sequence of KLK-L5 with members of the kalhkrein multigene family (See SEQ ID NOs 78-81, 83, 84) Dashes represent gaps to bring the sequences to better alignment
  • the residues of the catalytic triad are represented by bold letters, and the 29 invariant serine protease residues are marked with (•)
  • the cysteine residues are marked by ( ⁇ )
  • conserveed areas are highlighted in grey
  • the predicted cleavage sites in signal peptide are indicated by (4)
  • the dotted area represents the kalhkrein loop sequence
  • a vertical arrow marks the trypsin like cleavage site
  • Figure 37 is a dendrogram of the predicted phylogenetic tree for some serine proteases and other kalhkrein proteins Neighbor-joining/UPGMA method was used to align KLK-L5 with other serine proteases and members of the kalhkrein gene family The tree grouped the classical kailikreins (hKl, hK2, and PSA) together and aligned the KLK-L5 protein in one group with NES 1 and neuropsin Other serine proteases were aligned in different groups
  • Figure 38 shows tissue expression of the KLK-L5 gene as determined by RT-PCR
  • the upper band (905 base pairs, bp) is the classical form (see Figure 32, the middle (776 bp) the related protein- 1 , and the lower band (644 bp) the related prote ⁇ n-2
  • the primers used were L5-F2 and L5-R2, as shown in Table 17
  • Figure 39 shows hormonal regulation of the KLK-L5 gene in the LnCaP prostatic carcinoma cell line, BT-474 and T-47D breast carcinoma cell lines Steroids were at 10 8 M final concentration Actin (not regulated by steroid hoi mones) was used as a control gene Note detection ot three isotorms only in LNCaP
  • Figure 40 shows the expression of the KLK-L5 gene in breast cancer ( 1-17) and normal ( 18) tissues Note complete absence of expression in 12 cancer tissues.
  • Figure 38 Figure 41 shows the full structure of a KLK-L6 nucleic acid molecule
  • Figure 42 is a plot of hydrophobicity and hydrophilicity of KLK-L6 protein compared to prostate specific antigen (PSA).
  • Figure 43 shows an alignment of the deduced amino acid sequence of KLK-L6 with members of the kallikrem multigene family. (See SEQ. ID. NOs. 78-81, 83, 84). Dashes represent gaps to bring the sequences to better alignment
  • Figure 44 is a dendrogram of the predicted phylogenetic tree for some serine proteases and other kalhkrein proteins. Neighbor-joining UPGMA method was used to align KLK-L6 with other serine proteases and members of the kalhkrein gene family. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature.
  • nucleic Acid Molecules of the invention provides an isolated nucleic acid molecule having a sequence encoding a KLK-L Protein.
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized.
  • An "isolated” nucleic acid may also be free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) from which the nucleic acid is derived
  • nucleic acid is intended to include DNA and
  • RNA can be either double stranded or single stranded
  • a nucleic acid molecule encodes a KLK-L Protein comprising an amino acid sequence as shown in SEQ.ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, preferably a nucleic acid molecule comprising a nucleic acid sequence as shown in SEQ.ID.NO. 1, 13, 21, 43, 56, or 65.
  • the invention includes nucleic acid sequences complementary to a nucleic acid encoding a KLK-L
  • Protein comprising an amino acid sequence as shown in SEQ.ID.NO. 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67, preferably the nucleic acid sequences complementary to a full nucleic acid sequence shown in SEQ.ID NO.
  • the invention includes nucleic acid molecules having substantial sequence identity or homology to nucleic acid sequences of the invention or encoding proteins having substantial identity or similarity to the amino acid sequence shown in in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • the nucleic acids have substantial sequence identity for example at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% nucleic acid identity, more preferably 90% nucleic acid identity, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity "Identity" as known in the art and used herein, is a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences It also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequence
  • nucleic acid molecules encoding a KLK-L Protein and having a sequence which differs from a nucleic acid sequence of the invention due to degeneracy in the genetic code are also within the scope of the invention
  • nucleic acids encode functionally equivalent proteins (e g , a KLK-L Protein) but differ in sequence from the sequence of a KLK-L Protein due to degeneracy in the genetic code
  • DNA sequence polymorphisms within the nucleotide sequence of a KLK-L Protein may result in silent mutations which do not affect the ammo acid sequence Variations in one or more nucleotides may exist among individuals within a population due to natural allelic variation Any and all such nucleic acid variations are within the scope of the invention
  • DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of a KLK-L Protein These amino acid polymorphisms are also within the scope of the present invention
  • nucleic acid molecule which hybridizes under stringent conditions, preferably high stringency conditions to a nucleic acid molecule which comprises a sequence which encodes a KLK-L Protein having an amino acid sequence shown in SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1989), 6 3 1-6 3 6
  • 6 0 x sodium chloride/sodium citrate (SSC) at about 45°C, tollowed by a wash of 2 0 x SSC at 50°C may be employed
  • the stringency may be selected based on the conditions used in the wash step
  • the salt concentration in the wash step can be selected from a high stringency of about 0 2 x SSC at 50°C
  • the temperature in the wash step can be at
  • nucleic acid molecules of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of a nucleic acid sequence of the invention The labeled nucleic acid probe is used to screen an appropriate DNA library (e g a cDNA or genomic DNA library)
  • a cDNA library can be used to isolate a cDNA encoding a KLK-L Related Protein by screening the library with the labeled probe using standard techniques
  • a genomic DNA library can be similarly screened to isolate a genomic clone
  • An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a KLK-L Related Protein using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA It is possible to design synthetic oligonucleotide primers from the nucleotide sequence of the invention for use in PCR
  • a nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques
  • the nucleic acid so amplified can be cloned into an appropriate vector and characte ⁇ zed by DNA sequence analysis cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al , Biochemistry,
  • cDNA is then synthesized from the mRNA using reverse transc ⁇ ptase (for example, Moloney MLV reverse transc ⁇ ptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transc ⁇ ptase available from Seikagaku America, Inc , St Louis, FL)
  • reverse transc ⁇ ptase for example, Moloney MLV reverse transc ⁇ ptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transc ⁇ ptase available from Seikagaku America, Inc , St Louis, FL
  • RNA nucleic acid molecule of the invention which is RNA
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e g a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques
  • Nucleic acid molecules of the invention may be chemically synthesized using standard techniques Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid- phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e g , Itakura et al U S Patent No 4,598,049, Caruthers et al U S Patent No 4,
  • the initiation codon and untranslated sequences of a KLK-L Related Protein may be determined using computer software designed for the purpose, such as PC/Gene (IntelhGenetics Inc , Calif )
  • the intron-exon structure and the transcription regulatory sequences of a gene encoding a KLK-L Related Protein may be confirmed by using a nucleic acid molecule of the invention encoding a KLK-L Related Protein to probe a genomic DNA clone library
  • Regulatory elements can be identified using standard techniques
  • the function of the elements can be confirmed by using these elements to express a reporter gene such as the lacZ gene which is operatively linked to the elements
  • These constructs may be introduced into cultured cells using conventional procedures or into non-human transgenic animal models In addition to identifying regulatory elements in DNA, such constructs may also be used to identify nuclear proteins interacting with the elements, using techniques known in the art
  • the nucleic acid molecules isolated using the methods described herein are mutant klk-l gene alleles
  • the mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of for example, cancer (e g breast, testicular, brain, colon, and prostate cancer)
  • Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein
  • a cDNA of a mutant klk-l gene may be isolated using PCR as desc ⁇ bed herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutat ⁇ on(s) responsible for the loss or alteration of function of the mutant gene product
  • a genomic library can also be constructed using DNA from an individual suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from tissue known, or suspected to express the mutant allele
  • a nucleic acid encoding a normal klk-l gene or any suitable fragment thereof may
  • sequence of a nucleic acid molecule of the invention, or a fragment of the molecule may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule
  • An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art 2.
  • Proteins of the Invention An amino acid sequence ot a KLK L Protein comprises a sequence as shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • the proteins of the present invention include truncations of a KLK-L Protein, analogs of a KLK-L Protein, and proteins having sequence identity or similarity to a KLK-L Protein, and truncations thereof as described herein (I e included in KLK-L Related Proteins)
  • Truncated proteins may comprise peptides of between 3 and 70 amino acid residues, ranging in size from a t ⁇ peptide to a 70 mer polypeptide
  • the truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end
  • the truncated proteins may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end
  • the proteins of the invention may also include analogs of a KLK-L Protein, and/or truncations thereof as described herein, which may include, but are not limited to a KLK-L Protein, containing one or more amino acid substitutions, insertions, and/or deletions Amino acid substitutions
  • One or more amino acid insertions may be introduced into a KLK-L Protein Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length
  • Deletions may consist of the removal of one or more amino acids, or discrete portions from a KLK-L Protein sequence
  • the deleted amino acids may or may not be contiguous
  • the lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 20 to 40 amino acids
  • the proteins of the invention include proteins with sequence identity or similarity to a KLK-L
  • KLK-L Proteins include proteins whose amino acid sequences are comprised of the amino acid sequences of KLK-L Protein regions from other species that hybridize under selected hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a KLK-L Protein These proteins will generally have the same regions which are characteristic of a KLK-L Protein
  • a protein will have substantial sequence identity for example, about 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% identity, preferably 90% identity, more preferably at least 95%, 96%, 97%, 98%, or 99% identity, and most preferably 98% identity with an amino acid sequence shown in Tables 1 to 5 or SEQ ID NO 2, 3, 14, 22, 23, 44, 45, 57, 58, 59, 60, 66, or 67
  • a percent amino acid sequence homology, similarity or identity is calculated as the percentage ot aligned amino acids that match the reference sequence using known methods as described herein
  • the invention also contemplates isoforms of the proteins of the invention
  • An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure
  • Isoforms contemplated by the present invention preferably have the same properties as a protein of the invention as described herein
  • the present invention also includes KLK-L Related Proteins conjugated with a selected protein, or a marker protein (see below) to produce fusion proteins Additionally, immunogenic portions of a KLK- L Protein and a KLK-L Protein Related Protein are within the scope of the invention
  • a KLK-L Related Protein of the invention may be prepared using recombinant DNA methods Accordingly, the nucleic acid molecules of the present invention having a sequence which encodes a KLK- L Related Protein of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e g replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used
  • the invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology Methods m Enzymology 185, Academic Press, San Diego, CA (1990) Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art
  • the necessary regulatory sequences may be supplied by the native KLK-L Protein and/or its flanking regions
  • the invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of
  • the recombinant expression vectors of the invention may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention
  • marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG
  • the markers can be introduced on a separate vector from the nucleic acid of interest
  • the lecombinant expiession vectors may also contain genes which encode a fusion moiety which provides increased expression ot the recombinant protein, increased solubility of the recombinant protein, and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification
  • a proteolytic cleavage site may be added to the
  • Transformant host cells include host cells which have been transformed or transfected with a recombinant expression vector of the invention
  • the terms "transformed with”, “transfected with”, “transformation” and “transfection” encompass the introduction of a nucleic acid (e g a vector) into a cell by one of many standard techniques
  • Prokaryotic cells can be transformed with a nucleic acid by, for example, electroporation or calcium-chloride mediated transformation
  • a nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, pofectin, electroporation or microinjection Suitable methods for transforming and transfecting host cells can be found in Sambrook et al (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks Suitable host cells include a wide variety of prokaryotic
  • a host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e g glycosylation or phosphorylation) and processes (e g cleaves) the protein in a desired fashion
  • Host systems or cell lines may be selected which have specific and characteristic mechanisms for post-translational processing and modification of proteins
  • eukaryotic host cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38 may be used for long-term high-yield stable expression of the protein, cell lines and host systems which stably express the gene product may be engineered
  • Host cells and in particular cell lines produced using the methods desc ⁇ bed herein may be particularly useful in screening and evaluating compounds that modulate the activity of a KLK-L Related Protein
  • the proteins of the invention may also be expressed in non-human transgenic animals including but not limited to mice, rats, rabbits, guinea pigs, micro-pigs, goats, sheep, pigs, non-human primates (e g baboons, monkeys, and chimpanzees) [see Hammer et al (Nature 315 680-683, 1985), Palmiter et al
  • the transgene may be integrated as a single transgene or in concatamers
  • the transgene may be selectively introduced into and activated in specific cell types (See for example, Lasko et al, 1992 Proc. Natl. Acad. Sci. USA 89: 6236).
  • the transgene may be integrated into the chromosomal site of the endogenous gene by gene targeting.
  • the transgene may be selectively introduced into a particular cell type inactivating the endogenous gene in that cell type (See Gu et al Science 265: 103-106).
  • the expression of a recombinant KLK-L Related Protein in a transgenic animal may be assayed using standard techniques. Initial screening may be conducted by Southern Blot analysis, or PCR methods to analyze whether the transgene has been integrated. The level of mRNA expression in the tissues of transgenic animals may also be assessed using techniques including Northern blot analysis of tissue samples, in situ hybridization, and RT-PCR. Tissue may also be evaluated lmmunocytochemically using antibodies against KLK-L Protein.
  • Proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Mer ⁇ field, 1964, J. Am. Chem. Assoc. 85:2149- 2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed E Wansch, Vol. 15 I and II, Thieme, Stuttgart).
  • N-terminal or C-terminal fusion proteins comprising a KLK-L Related Protein of the invention conjugated with other molecules, such as proteins, may be prepared by fusing, through recombinant techniques, the N-terminal or C-termmal of a KLK-L Related Protein, and the sequence of a selected protein or marker protein with a desired biological function.
  • the resultant fusion proteins contain KLK-L
  • proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc 3.
  • Antibodies KLK-L Related Proteins of the invention can be used to prepare antibodies specific for the proteins. Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein An unconserved region of the protein is one that does not have substantial sequence homology to other proteins. A region from a conserved region such as a well-characte ⁇ zed domain can also be used to prepare an antibody to a conserved region of a KLK-L Related Protein. Antibodies having specificity for a KLK-L Related Protein may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein
  • the invention can employ intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g. a Fab, (Fab) 2 fragment, or Fab expression library fragments and epitope-binding fragments theieof), an antibody heavy chain, and antibody light chain, a genetically engineered single chain Fv molecule (Ladner et al, U S Pat No 4,946,778), or a chimenc antibody, for example, an antibody which contains the binding specificity ot a mu ⁇ ne antibody, but in which the remaining portions are of human origin
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras may be prepared using methods known to those skilled in the art
  • the nucleic acid molecules, KLK-L Related Proteins, and antibodies of the invention may be used in the prognostic and diagnostic evaluation of cancer (e g breast, testicular, and prostate cancer) or other conditions, and the identification of subjects with a predisposition to cancer (Section 4 1 1 and 4 1 2)
  • Methods for detecting nucleic acid molecules and KLK-L Related Proteins of the invention can be used to monitor conditions including cancer, by detecting KLK-L Related Proteins and nucleic acid molecules encoding KLK-L Related Proteins
  • the applications of the present invention also include methods for the identification of compounds that modulate the biological activity of KLK-L or KLK-L Related Proteins (Section 4 2)
  • the compounds, antibodies etc may be used for the treatment of cancer (Section 4 3) 4.1 Diagnostic Methods
  • a variety of methods can be used to monitor conditions including cancer, by detecting KLK-L Related Proteins and nucleic acid molecules encoding KLK-L Related Proteins.
  • the methods desc ⁇ bed herein may be performed by utilizing pre-packaged diagnostic kits comprising at least one specific KLK-L nucleic acid or antibody described herein, which may be conveniently used, e g , in clinical settings, to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a disorder
  • Nucleic acid-based detection techniques are described, below, in Section 4 1 1
  • Peptide detection techniques are described, below, in Section 4 1 2
  • the samples that may be analyzed using the methods of the invention include those which are known or suspected to express KLK-L or contain KLK-L Related Proteins
  • the samples may be derived from a patient or a cell culture, and include but are not limited to biological fluids, tissue extracts, treshly harvested cells, and lysates ot cells which have been incubated in cell cultures
  • Oligonucleotides or longer fragments derived trom any of the nucleic acid molecules of the invention may be used as targets in a microarray
  • the microarray can be used to simultaneously monitor the expression levels of large numbers of genes and to identify genetic variants, mutations, and polymorphisms
  • the information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents
  • nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences of the invention in samples Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding at least
  • the nucleic acid probes may be used to detect genes, preferably in human cells, that encode KLK-L Related Proteins
  • the nucleotide probes may also be useful in the diagnosis of cancer, in monitoring the progression of cancer, or monitoring a therapeutic treatment
  • the probe may be used in hybridization techniques to detect genes that encode KLK-L Related
  • the technique generally involves contacting and incubating nucleic acids (e g recombinant DNA molecules, cloned genes) obtained from a sample from a patient or other cellular source with a probe of the present invention under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected
  • nucleic acids e g recombinant DNA molecules, cloned genes
  • the detection of nucleic acid molecules of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art Suitable primers can be routinely designed by one ot skill in the art
  • Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving klk I structure, including point mutations, insertions, deletions, and chromosomal rearrangements
  • direct sequencing single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and oligonucleotide hybridization may be utilized
  • Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to the mutations in a klk-l gene
  • the polymorphisms may be used to identify individuals in families that are likely to carry mutations If a polymorphism exhibits linkage disequalib ⁇ um with mutations in a klk-l gene, it can also be used to screen for individuals in the general population likely to carry mutations
  • Polymorphisms which may be used include restriction fragment length polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat polymorphisms (SSLPs)
  • a probe of the invention may be used to directly identify RFLPs
  • a probe or primer of the invention can additionally be used to isolate genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids
  • genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids
  • the DNA in the clones can be screened for SSLPs using hybridization or sequencing procedures
  • Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of klk-l expression
  • RNA may be isolated from a cell type or tissue known to express klk-l and tested utilizing the hybridization (e g standard Northern analyses) or PCR techniques referred to herein
  • the techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing
  • the techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcripts detected in normal individuals relative to those individuals exhibiting cancer symptoms or other disease conditions
  • the primers and probes may be used in the above desc ⁇ bed methods in situ l e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections
  • Antibodies specifically reactive with a KLK-L Related Protein, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect KLK-L Related Proteins in various samples (e g biological materials) They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of KLK-L Related Proteins expression, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of a KLK-L Related Protein Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on cancer, and other conditions In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies The antibodies of the invention may also be used in vitro to determine the level of KLK-L expression in cells genetically engineered to produce a KLK-L Related Protein
  • the antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a KLK-L Related Protein and the antibodies Examples of such assays are radioimmunoassays, enzyme immunoassays (e g ELISA), lmmunofluorescence, immunoprecipitation, latex agglutination, hemagglutmation, and histochemi il tests
  • the antibodies may be used to detect and quantity KLK-L Related Proteins in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states
  • the antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a KLK-L Related Protein, to localize it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a KLK-L Related Protein
  • an antibody of the invention may be labeled with a detectable substance and a KLK-L Related Protein may be localised m tissues and cells based upon the presence of the detectable substance
  • detectable substances include, but are not limited to, the following radioisotopes (e g , 3 H, 14 C, 35 S, 125 I, 131 I), fluorescent labels (e g , FITC, rhodamine, lanthanide phosphors), luminescent labels such as lurrunol, enzymatic labels (e g , horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylchohnesterase), biotinyl groups (which can be detected by marked avidin e g , streptavidin containing a fluorescent marker or
  • a KLK-L Related Protein may be localized by radioautography
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains 4.2 Methods for Identifying or Evaluating Substances/Compounds
  • the methods described herein are designed to identify substances that modulate the biological activity of a KLK-L Related Protein including substances that bind to KLK-L Related Proteins, or bind to other proteins that interact with a KLK-L Related Protein, to compounds that interfere with, or enhance the interaction of a KLK-L Related Protein and substances that bind to the KLK-L Related Protein or other proteins that interact with a KLK-L Related Protein Methods are also utilized that identify compounds that bind to KLK L regulatory sequences
  • the substances and compounds identified using the methods ot the invention include but aie not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members ot random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e g polyclonal, monoclonal, humanized, anti-idiotypic,
  • a substance that associates with a polypeptide of the invention may be an agonist or antagonist of the biological or immunological activity of a polypeptide of the invention
  • the term "agonist' refers to a molecule that increases the amount of, or prolongs the duration of, the activity of the polypeptide
  • the term "antagonist” refers to a molecule which decreases the biological or immunological activity of the polypeptide
  • Agonists and antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that associate with a polypeptide of the invention
  • Substances which can bind with a KLK-L Related Protein may be identified by reacting a KLK-L Related Protein with a test substance which potentially binds to a KLK-L Related Protein, under conditions which permit the formation of substance-KLK-L Related Protein complexes and removing and/or detecting the complexes
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof
  • isolation techniques for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof
  • antibody against KLK-L Related Protein or the substance, or labeled KLK-L Related Protein, or a labeled substance may be utilized.
  • the antibodies, proteins, or substances may be labeled with a detectable substance as described above
  • a KLK-L Related Protein, or the substance used in the method of the invention may be insolubilized
  • a KLK-L Related Protein, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, lon- exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc
  • the insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical ethods, toi example, cyanogen bromide coupling
  • the invention also contemplates a method tor evaluating a compound for its ability to modulate the biological activity of a KLK-L Related Protein ot the invention, by assaying for an agonist or antagonist (1 e enhancer or inhibitor) of the binding of a KLK-L Related Protein with a substance which binds with a KLK-L Related Protein
  • the basic method for evaluating if a compound is an agonist or antagonist of the binding of a KLK-L Related Protein and a substance that binds to the protein is to prepare a reaction mixture containing the KLK-L Related Protein and the substance under conditions which permit the formation of substance-KLK-L Related Protein complexes, in the presence of a test compound
  • the test compound may be initially added to the mixture, or may be added subsequent to the addition of the KLK-L Related Protein and substance Control reaction mixtures without the test compound or with a placebo are also prepared
  • the formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound
  • agonists and antagonists l e inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or alloste ⁇ c sites
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of KLK-L Related Protein with a substance which is capable of binding to the KLK-L Related Protein
  • the invention may be used to assay for a compound that competes for the same binding site of a KLK-L Related Protein
  • the invention also contemplates methods for identifying compounds that bind to proteins that interact with a KLK-L Related Protein Protein-protein interactions may be identified using conventional methods such as co-immunoprecipitation, crosshnking and co-purification through gradients or chromatographic columns Methods may also be employed that result in the simultaneous identification of genes which encode proteins interacting with a KLK-L Related Protein These methods include probing expression libraries with labeled KLK-L Related Protein
  • Two-hybrid systems may also be used to detect protein interactions in vivo
  • plasmids are constructed that encode two hybrid proteins
  • a first hybrid protein consists of the DNA-binding domain of a transcription activator protein fused to a KLK-L Related Protein
  • the second hybrid protein consists of the transcription activator protein's activator domain fused to an unknown protein encoded by a cDNA which has been recombined into the plasmid as part of a cDNA library
  • the plasmids are transformed into a strain of yeast (e g S cerevisiae) that contains a reporter gene (e g lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the transcription activator's binding site
  • yeast e g S cerevisiae
  • a reporter gene e g lacZ, luciferase, alkaline phosphatase, horseradish peroxidase
  • fusion proteins may be used in the above-described methods
  • KLK-L Related Proteins fused to a glutathione S-transferase may be used in the methods
  • the reagents suitable for applying the methods of the invention to evaluate compounds that modulate a KLK-L Related Protein may be packaged into convenient kits providing the necessary materials packaged into suitable containers
  • the kits may also include suitable supports useful in performing the methods of the invention 4.3 Compositions and Treatments
  • the proteins of the invention, substances or compounds identified by the methods desc ⁇ bed herein, antibodies, and antisense nucleic acid molecules of the invention may be used for modulating the biological activity of a KLK-L Related Protein, and they may be used in the treatment of conditions such as cancer (e g prostate, testicular, brain, uterine, thymus, ovarian, colon, ovarian, or breast cancer)
  • the substances, antibodies, peptides, and compounds may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in viv
  • the active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated in a material to protect the substance from the action of enzymes, acids and other natural conditions that may inactivate the substance
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa , USA 1985) On this basis, the compositions include, albeit not exclusively, solutions of the active substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and lso-osmotic with the physiological fluids Based upon their homology to genes encoding kalhkrein, nucleic acid molecules ot the invention may be also useful in the treatment ot conditions such as hypertension, cardiac hypertrophy, arthritis, inflammatory disorders, neurological disorders, and blood clotting disorders
  • Vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used to deliver nucleic acid molecules to a targeted organ, tissue, or cell population Methods well known to those skilled in the art may be used to construct recombinant vectors which will express antisense nucleic acid molecules of the invention (See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra))
  • nucleic acid molecules comprising full length cDNA sequences and/or their regulatory elements enable a skilled artisan to use sequences encoding a protein of the invention as an investigative tool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol 13 98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60 631-652) regulation of gene function
  • sense or antisense o gomers, or larger fragments can be designed from various locations along the coding or control regions
  • Genes encoding a protein of the invention can be turned off by transfectmg a cell or tissue with vectors which express high levels of a desired KLK-L-encoding fragment
  • Such constructs can inundate cells with untranslatable sense or antisense sequences Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases Modifications of gene expression can be obtained by designing antisense molecules,
  • Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of RNA Ribozymes act by sequence-specific hybridization of the ⁇ bozyme molecule to complementary target RNA, followed by endonucleolytic cleavage
  • the invention therefore contemplates engineered hammerhead motif ⁇ bozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a protein of the invention
  • ⁇ bozyme cleavage sites within any potential RNA target may initially be identified by scanning the target molecule for ⁇ bozyme cleavage sites which include the following sequences, GUA,
  • RNA sequences of between 15 and 20 ⁇ bonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable
  • the suitability of candidate targets may also be detei mined by testing accessibility to hybridization with complementary oligonucleotides using ⁇ bonuclease piotection assays
  • Methods tor introducing vectors into cells or tissues include those methods discussed herein and which are suitable tor m vivo, m vitro and ex vivo therapy
  • vectors may be introduced into stem cells obtained from a patient and clonally propagated for autologous transplant into the same patient (See U S Pat Nos 5,399,493 and 5,437,994) Delivery by transfection and by liposome are well known in the art
  • nucleic acid molecules disclosed herein may also be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions
  • the invention also provides methods for studying the function of a polypeptide of the invention
  • Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of a nucleic acid molecule or gene of the invention may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations in the gene
  • a recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue, or animal
  • Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation
  • a recombinant gene may also be engineered to contain an insertion mutation that inactivates the gene
  • Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc
  • Cells lacking an intact gene may then be identified, for example by Southern blotting, Northern Blotting, or by assaying for expression of the encoded polypeptide using the methods described herein
  • Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in a polypeptide of the invention
  • Germlme transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embryos, in vitro, transferring the resulting blastocysts into recipient females and, generating germhne transmission of the resulting aggregation chimeras
  • Such a mutant animal may be used to define specific
  • the invention provides a transgenic non-human mammal all of whose germ cells and somatic cells contain a recombinant expression vector that inactivates or alters a gene encoding a KLK- L Related Protein resulting in a KLK-L Related Protein associated pathology Further the invention provides a transgenic non-human mammal which doe not express a KLK-L Related Protein of the invention. In an embodiment, the invention provides a transgenic non-human mammal which doe not express a KLK-L Related Protein of the invention resulting in a KLK-L Related Protein associated pathology
  • a KLK-L Related Protein pathology refers to a phenotype observed for a KLK-L Related Protein homozygous mutant
  • a transgenic non-human animal includes but is not limited to mouse, rat, rabbit, sheep, hamster, dog, guinea pig, micro-pig, pig, cat, goat, and non-human primates, preferably mouse
  • the invention also provides a transgenic non-human animal assay system which provides a model system for testing for an agent that reduces or inhibits a pathology associated with an KLK-L Related Protein, preferably a KLK-L Related Protein associated pathology, comprising
  • step (b) determining whether said agent reduces or inhibits the pathology (e g KLK-L Related Protein associated pathology) in the transgenic non-human animal relative to a transgenic non-human animal of step (a) which has not been administered the agent
  • the agent may be useful in the treatment and prophylaxis of conditions such as cancer as discussed herein
  • the agents may also be incorporated in a pharmaceutical composition as described herein
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics
  • the therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED 50 /LD 50 ratio
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred
  • the Lawrence Livermore National Laboratory participates in the sequencing of the human genome project and focuses on sequencing chromosome 19 Large sequencing information on this chromosome is available at the website of the Lawrence Livermore National Laboratory (http //www- bio llnl gov/genome/gemnome html)
  • BAC 288H1 and BAC 76F7 Two clones which were positive for the Zyme gene (clones BAC 288H1 and BAC 76F7) These BACs were further analyzed by PCR and primers specific for PSA, NESl, KLKl and KLK2 These analyses indicated that both BACs were positive for Zyme, PSA and
  • KLKl, KLK2 and KLK3 are considered to represent the human kalhkrein gene family (9)
  • the work described herein provides strong evidence that a large number of kallikrein-hke genes are clustered within a 300Kb region around chromosome 19q l3 2 - ql3 4
  • the three established human kailikreins KLKl, KLK2, KLK3), Zyme and NESl, as well as the stratum corneum chymotrypticn enzyme, neuropsin, and TLSP (trypsin-like serine protease) and another five new genes , KLK-Ll to KLK-L5, may constitute a large gene family This will bring the total number of kalhkrein or kallikrein-hke genes in this region of chromosome 19 to thirteen
  • the human stratum corneum chymotryptic enzyme (19), neuropsin (20) and trypsin-hke serine protease (TLSP) (21) are three previously characterized genes which have many structural similarities with the kailikreins and other members of the serine protease family However, they have not been mapped in the past Their precise mapping in the region ot the kalhkrein gene family indicates that these three genes, along with the ones that were newly identified, or are already known, constitute a family that likely originated by duplication of an ancestral gene The relative localization of all these genes is depicted in Figure 1
  • Kalhkrem genes are a subfamily of serine proteases, traditionally characterized by their ability to liberate lysyl-bradykinin (kalhdin) from kininogen (18) More recently, however, a new, structural concept has emerged to describe kailikreins From accumulated sequence data, it is now clear that the mouse has many genes with high homology to kalhkrein coding sequences (19-20) Richard and co-workers have contributed to the concept of a " kal krem multigene family" to refer to these genes (21-22) This definition is not based much on specific enzymatic function of the gene product, but more on its sequence homology and their close linkage on mouse chromosome 7 In humans, only KLKl meets the functional definition of a kalhkrein KLK2 has trypsin-hke enzymatic activity and KLK3 (PSA) has very weak chymotrypsin-Iike enzymatic activity These activities of KLK2 and KLK3 are not known to
  • the breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD. BT-474 cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic tlasks, to near contluency The cells were then ahquoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 8 M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction
  • RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as desc ⁇ bed above for the tissue culture experiments and used for PCR reactions with the primers described in Table 8 (SEQ ID Nos 5-12) Tissue cDNAs were amplified at various dilutions Cloning and sequencing of the PCR products
  • the exon prediction strategy of the 300Kb DNA sequences around chromosome 19ql3 3 - ql3 4 identified a novel gene with a structure reminiscent of a serine protease
  • the major features of this gene were its homology, at the amino acid and DNA level, with other human kalhkrein genes, the conservation of the catalytic triad (histidine, aspartic acid, and serine), the number of exons and the complete conservation of the intron phases EST sequence homology search
  • the coding sequence of the klk-L2 gene is shown in SEQ ID NO 1 and GenBAnk Accession # AF135023
  • the exons of the gene are as follows exon 1 (939-999), exon 2 (2263-2425), exon 3 (2847- 3097), exon 4 (3181-3317), and exon 5 (4588-4740)
  • the amino acid sequence of KLK-L2 proteins are shown in SEQ ID Nos 2 and 3 Mapping and chromosomal localization of prostase /KLK-Ll gene
  • the tissue expression of prostase/KLK-Ll is summarized in Table 9 and Figure 3
  • This protease is primarily expressed in the prostate, testis, adrenals, uterus, thyroid, colon, central nervous system and mammary tissues, and, at much lower levels in other tissues
  • the specificity of the RT-PCR procedure was verified for prostase/KLK-Ll by cloning the PCR products from mammary, testicular and prostate tissues and sequencing them
  • One example with mammary tissue is shown in Figure 4 All cloned PCR products were identical in sequence to the cDNA sequence reported for the prostase/KLK-Ll Hormonal regulation of the prostase/KLK-Ll gene
  • the steroid hormone receptor-positive breast carcinoma cell line BT-474 was used as a model system to evaluate whether prostase/KLK-Ll expression is under steroid hormone regulation
  • the controls worked as expected I e , actin positivity without hormonal regulation in all cDNAs, only
  • the KLK3 gene encodes for PSA, a protein that currently represents the best tumor marker available (24) Since in rodents there are so many kalhkrein genes, the restriction of this family to only 3 genes in humans was somewhat surprising More recently, new candidate kalhkrein genes in humans have been discovered, including NESl (13) and zyme/protease M neurosin (10-12)
  • the known kailikreins and the newly discovered kalhkrein-hke genes share the following similarities (a) they encode serine proteases (b) they have five coding exons (c) they share significant DNA and protein homologies with each other (d) they map in the same locus on chromosome 19ql3 3-ql3 4, a region that is structurally similar to an area on mouse chromosome 7, where all the mouse kalhkrein genes are localized (e) they appear to be regulated by steroid hormones Prostase/KLK-Ll is a member of the same family since these common characteristics are also
  • the sensitive RT-PCR protocol reveals that the KLK-Ll enzyme is expressed m prostatic tissue and it is also expressed in significant amounts in other tissues, including testis, female mammary gland, adrenals, uterus, thyroid, colon, brain, lung and salivary glands ( Figure 3 and Table 9)
  • the specificity of the RT-PCR primers was verified by sequencing the obtained PCR products, with one example shown in Figure 4 (SEQ ID NO 4)
  • Tissue culture studies with the breast carcinoma cell line BT-474 further confirm not only the ability of these cells to produce prostase/KLK-Ll but also its hormonal regulation (Figure 5)
  • An interesting theme is now developing involving the group of homologous genes on chromosome 19ql3 3(PSA, KLK2, prostase, zyme, and NESl)
  • the combined data suggest that all of them are expressed in prostate and breast tissues, and all of them are hormonally regulated All these genes may be part of a cascade pathway that plays a role in cell proliferation, differentiation or
  • prostase/KLK-Ll can be produced by these cells and that its expression is significantly up-regulated by androgens and progestins Based on information for other homologous genes in the area ( PSA, zyme, and NESl), prostase/KLK-Ll may be involved in the pathogenesis and/or progression of prostate, breast and possibly other cancers
  • PSA prostate carcinoma cell lines
  • Sequencing data of approximately 300Kb of nucleotides on chromosome 19ql3.3-ql3 4 was obtained from the web site of the Lawrence Livermore National Laboratory (LLNL) (http://www- bio.llnl.gov/genome/genome.html). This sequence was in the form of 9 contigs of different lengths.
  • a restriction analysis study of the available sequences was performed using the "WebCutter" computer program (httpV/www.firstmarket. com/cutter/cut2.html) and with the aid of the EcoRI restriction map of this area (also available from the LLNL web site) an almost contiguous stretch of genomic sequences was constructed.
  • the KLK-L2 gene is formed of 5 coding exons and 4 intervening introns, spanning an area of 9,349 bp of genomic sequence on chromosome 19ql3 3-ql3 4
  • the lengths of the exons are 73, 262, 257,134, and 156 bp, respectively
  • the intron/exon splice sites (mGT AGm) and their flanking sequences are closely related to the consensus splicing sites (-mGTAAGT CAGm-) (32)
  • the presumptive protein coding region of the KLK-L2 gene is formed of 879 bp nucleotide sequence encoding a deduced 293-am ⁇ no acid polypeptide with a predicted molecular weight of 32 KDa
  • There are two potential translation initiation codons (ATG) at positions 1 and 25 of the predicted first exon (numbers refer to SEQ ID NO .
  • KLK-L2 will possess a trypsin-like cleavage pattern like most of the other kailikreins (e g , KLKl , KLK2, TLSP, neuropsin, zyme, prostase, and EMSP) but different from PSA which has a serine (S) residue in the corresponding position, and is known to have a chymotrypsin like activity ( Figure 9)
  • the dotted region in Figure 9 indicates an 11-am ⁇ no acid loop characteristic of the classical kailikreins (PSA, KLKl, and KLK2) but not found in KLK-L2 or other members of the kallikrein-hke gene family (11) Homology with the kalhkrein multi-gene family
  • the mature 227-am ⁇ no acid sequence of the predicted protein was aligned against the GenBank database and the known kailikreins using the "BLASTP” and "BLAST 2 sequence” programs KLK-L2 is found to have 54% amino acid sequence identity and 68% similarity with the enamel matrix serine proteinase 1 (EMSPl ) gene, 50% identity with both trypsin like serine protease (TLSP) and neuropsin genes and 47%, 46%, and 42% identity with trypsinogen, zyme, and PSA genes, respectively
  • the multiple alignment study shows that the typical catalytic triad of serine proteases is conserved in the KLK-L2 gene (H 108 , D 153 , and S 245 ) and, as the case with all other kailikreins, a well conserved peptide motif is found around the amino acid residues of the catalytic triad [I e , histidine (WLLTAA/7C), se ⁇ ne(GD
  • the KLK-L2 gene is primarily expressed in the brain, mammary gland, and testis but lower levels of expression are found in many other tissues
  • the PCR products were cloned and sequenced Hormonal regulation of the KLK-L2 gene
  • a steroid hormone receptor positive breast cancer cell line (BT-474) was used as a model to verify whether the KLK-L2 gene is under steroid hormone regulation PSA was used as a control known to be upregulated by androgens and progestins and pS2 as an estrogen upregulated control
  • PSA steroid hormone receptor positive breast cancer cell line
  • KLK-L2 for kallikrem like gene 2
  • the KLK-L2 gene is composed of 5 coding exons and 4 intervening introns and, except for the second coding exon, the exon lengths are comparable to those of other members of the kalhkrein gene family ( Figure 6)
  • the exon-intron splice junctions were identified by comparing the genomic sequence with the EST sequence and were further confirmed by the conservation of the consensus splice sequence (-mGT AGm-) (32), and the fully conserved intron phases, as shown in Figure 6. Furthermore, the position of the catalytic triad residues in relation to the different exons is also conserved (Figure 6).
  • KLK-L2 is more functionally related to trypsin than to chymotrypsin (34).
  • the wide range of tissue expression of KLK-L2 should not be surprising since, by using the more sensitive RT-PCR technique instead of Northern blot analysis, many kalhkre genes were found to be expressed in a wide variety of tissues including salivary gland, kidney, pancreas, brain, and tissues of the reproductive system (uterus, mammary gland, ovary, and testis) (34). KLK-L2 is highly expressed in the brain.
  • each kalhkrein enzyme has one specific physiological substrate
  • Serine proteases encode protein cleaving enzymes that are involved in digestion, tissue remodeling, blood clotting etc., and many of the kalhkrein genes are synthesized as precursor proteins that must be activated by cleavage of the propeptide.
  • KLK-L2 The predicted trypsin-hke cleavage specificity of KLK-L2 makes it a candidate activator of other kailikreins or it may be involved in a "cascade" of enzymatic reactions similar to those found in fib ⁇ nolysis and blood clotting (36)
  • KLK-L2 a new member of the human kalhkrein gene family, KLK-L2 was characterized This gene is hormonally regulated and it is mostly expressed in the brain, mammary gland and testis. KLK- L2 may be useful as a tumor marker Example 4
  • RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as desu ibed below and used tor PCR amplification A pinner set (L3 FI and L3-R1) was used to identity the presence of the gene in tissues, and the reverse primer (L -R1) was used with another primer (L3-F2) to amplify and clone the full cDNA of the gene
  • L3 FI and L3-R1 was used to identity the presence of the gene in tissues
  • L3-R1 was used with another primer (L3-F2) to amplify and clone the full cDNA of the gene
  • RNA 2 ⁇ g was reverse-transcribed into first strand cDNA using the SuperscriptTM preamplification system (Gibco BRL, Gaithersburg, MD) The final volume was 20 ⁇ l
  • two gene-specific primers (L3-F1 and L3-R1) were designed (Table 13, SEQ ID Nos 9-12, 24- 26) and PCR was carried out in a reaction mixture containing 1 ⁇ l of cDNA, 10 mM T ⁇ s-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl 2 , 200 ⁇ M dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmphTaq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) on a Perkin- Elmer 9600 thermal cycler The cycling conditions were 94°C for 9 minutes, followed by 43 cycles of 94°C for
  • the breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then a quoted into 24-well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 8 M Cells stimulated with 100% ethanol were included as controls
  • the cells were cultured for 24 hours, then harvested for total RNA extraction by the T ⁇ zol method (Gibco- BRL) cDNA was prepared and amplified as described above Control genes (PSA, pS2, and actin) were amplified as previously described herein Cloning and sequencing of the PCR products. To verify the identity of the PCR products, they were cloned into the pCR 2 1-TOPO vector
  • the PCR product generated with primer set Z1S and Zl AS (Table 14, SEQ ID NOS 27-42), was purified and then labeled with 32 P by the random primer method (Sambrook, supra) and used as a probe to screen a human genomic DNA BAC library, spotted in duplicate on nylon membranes, for identification of positive clones
  • the filters were hybridized in 15% formamide, 500 mM Na 2 HP0 4 , 7% SDS, 1% BSA
  • KLKl human glandular kallikrem
  • KLK2 human glandular kallikrem
  • NESl normal epithelial cell-specific lgene
  • KLK-Ll KLK- L2 and zyme genes
  • gene-specific primers were designed for each of these genes (Table 14) and developed polymerase chain reaction (PCR)-based amplification protocols which allowed us to generate specific PCR products with genomic DNA as a template
  • PCR reactions were carried out as described above but by using an annealing/extension temperature of 65°C. Structure analysis studies.
  • the KLK-L3 gene is formed of 5 coding exons and 4 intervening introns, although, as with other kailikreins, the presence of further upstream untranslated exon(s) could not be ruled out (14, 28) All of the exon /intron splice sites conform to the consensus sequence for eukaryotic splice sites (32) The gene further follows strictly the common structural features of the human kalhkrein multigene family, as described below
  • the predicted protein-coding region of the gene is formed of 753 bp, encoding a deduced ammo acid polypeptide with a predicted molecular weight of 27 5 kDa
  • a potential translation initiation codon is found at position 28 of the predicted first exon (numbers of nucleotides refer to SEQ ID NO 21 or GenBAnk Accession # API 35026
  • This codon does not match well with the consensus Kozak sequence (33), however, it has a pu ⁇ ne at position (-3) which occurs in 97% of vertebrate mRNAs (43), and it is almost identical to the sequence of the zyme gene flanking the start codon
  • Nucleotides 6803-6808 (AGTAAA) closely resemble a consensus polyadenylation signal (44) and are followed by a stretch of 19 poly A nucleotides not found in genomic DNA, after a space of 14 nucleotides
  • the KLK-L3 gene is primarily expressed in thymus, testis, spinal cord, cerebellum, trachea, mammary gland, prostate, brain, salivary gland, ovary and skin (the latter two tissues are not shown in the figure) Lower levels of expression are seen m fetal brain, stomach, lung, thyroid, placenta, liver, small intestine, and bone marrow No expression was seen in uterus, heart, fetal liver, adrenal gland, colon, spleen, skeletal muscle, pancreas, and kidney In order to verify the RT-PCR specificity, representative PCR products were cloned and sequenced Figure 20 shows that KLK-L3 gene is regulated by steroid hormones in the human breast cancer cell line BT-474 DISCUSSION
  • kailikreins are characterized by their ability to liberate lysyl-bradykinin (kal din) tiom kininogen (2)
  • KLK2 and KLK3 are assigned to the same family based on the strong structural similarities of the genes and proteins and the close localization of these genes on the same chromosomal region (20)
  • Richards and co-workers introduced the concept of a "kalhkrein multigene family" in mice, to refer to these genes (20, 21)
  • This definition is not based much on the specific enzymatic function of the gene product, but more on its sequence homology and its close linkage on mouse chromosome 7 Irwin et al (27) proposed that the serine protease genes can be classified into five different groups according to intron position as discussed above The results indicate the presence of some more common structural features that are found in all kailikreins (including the
  • the chromosomal band 19ql3 is nonrandomly rearranged in a variety of human solid tumors including ovarian cancers (46), and the currently available data indicate that the kalhkrein gene locus is related to many malignancies At least three kalhkrein genes (PSA, zyme and NESl) are down regulated in breast cancer (10, 13, 47, 48), and NESl appears to be a novel tumor suppressor gene (29) Furthermore, PSA exhibits potent antiangiogenic activity (49) It is possible that some of these kailikreins are involved in a cascade pathway, similar to the coagulation or apoptotic process, whereby pro-forms of proteolytic enzymes are activated and then act upon downstream substrates Such activity was found for the KLK2 gene product which acts upon and activates pro PSA (50, 51)
  • the expanded human kalhkrein gene family has similar number of members as the rodent family of genes Some new compelling data have raised the possibility that at least some of these genes behave as tumor suppressors (29), as negative regulators of cell growth (52), as antiangiogenic (49) and apoptotic (53) molecules
  • the paramount diagnostic value of some members is also well-established (24, 54) For these reasons, it is important to check all members of this family of genes as potential diagnostic or prognostic markers or as candidate therapeutic targets
  • the newly identified KLK-L3 gene is expressed in many tissues, including skin, thymus, central nervous system, breast, prostate, and testis
  • the wide range of tissue expression of KLK-L3 should not be surprising since, by using the more sensitive RT-PCR technique, many kalhkrein genes were found to be expressed in a wide variety of tissues For example, PSA, KLK2, prostase/KLK-Ll, and KLK-L2 are now known to be expiessed in breast and many other tissues (30, 54)
  • KLK-L3 is regulated by steroid hormones but in a more complex fashion than PSA and KLK2 which are up-regulated by androgens and progestins (71).
  • PSA serotonins
  • KLK-L3 appears to be up-regulated by progestins > estrogens > androgens (Figure 20)
  • LLNL Lawrence Livermore National Laboratory
  • RNA isolated from 26 different human tissues was purchased from Clontech.
  • cDNA was prepared as described below, and used for PCR reactions with different sets of primers (Table 15, SEQ.IDNOs 46-55, 9-12) Tissue cD ⁇ As were amplified at various dilutions Breast cancer cell line and hormonal stimulation experiments
  • the breast cancer cell line BT-474 was purchased from the American Type Culture Collection (ATCC), Rockville, MD. Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency. The cells were then a quoted into 24- well tissue culture plates and cultured to 50% confluency.
  • the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum
  • various steroid hormones dissolved in 100% ethanol were added into the culture media, at a final concentration of 10 "8 M Cells stimulated with 100% ethanol were included as controls
  • the cells were cultured tot 24 houis, then harvested for mRNA extraction Reverse transcriptase polymerase chain reaction
  • PCR was carried out in a reaction mixture containing 1 ⁇ l of cD ⁇ A, 10 mM T ⁇ s-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 200 ⁇ M d ⁇ TPs (deoxynucleoside triphosphates), 150 ng of primers and 2.5 units of AmpliTaq Gold D ⁇ A polymerase (Roche Molecular Systems, Branchburg, ⁇ J, USA) on a Perkin-Elmer 9600 thermal cycler.
  • the cycling conditions were 94°C for 9 minutes to activate the Taq Gold D ⁇ A polymerase, followed by 43 cycles of 94°C for 30 s, 63°C for 1 minute and a final extension at 63°C for 10 min
  • Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. All primers for RT-PCR spanned at least 2 exons to avoid contamination by genomic D ⁇ A.
  • Normal breast tissues were obtained from women undergoing reduction mammoplasties.
  • Breast tumor tissues were obtained from female patients at participating hospitals of the Ontario Provincial Steroid Hormone Receptor Program The normal and tumor tissues were immediately frozen in liquid nitrogen after surgical resection and stored in this manner until extracted The tissues were pulverized with a hammer at dry ice temperature and RNA was extracted as described above, using T ⁇ zol reagent.
  • KLK-L4 is formed of five coding exons and four intervening introns, spanning an area of 8,905 bp of genomic sequence on chromosome 19ql3 3-ql3 4
  • the lengths of the coding regions are 52, 187, 269,137 and 189 bp, respectively
  • the predicted protein coding region of the gene is formed of 831 bp, encoding a deduced
  • Kb area of interest [the human kalhkrein gene family locus], enabled precise localization of all known genes and to determine the direction of transcription, as shown by the arrows in Figure 28
  • the PSA gene lies between KLKl and KLK2 genes and is separated by 13, 319 base pairs (bp) from KLK2, and both genes are transcribed in the same direction (centromere to telomere) All other kallikrein-hke genes are transcribed in the opposite direction
  • KLK-L4 is 13 kb centromeric from KLK-L6 [SEQ ID NO 65], and
  • the KLK-L4 gene is primarily expressed in mammary gland, prostate, salivary gland and testis, but, as is the case with other kailikreins, lower levels of expression are found in many other tissues
  • the PCR products were cloned and sequenced
  • a steroid hormone receptor-positive breast cancer cell line (BT-474) was used as a model, to verify whether the KLK-L4 gene is under steroid hormone regulation PSA was used as a control gene, known to be up-regulated by androgens and progestins and pS2 as an estrogen up-regulated control gene in the same cell line
  • Preliminary results indicate that KLK-L4 is up-regulated by progestins and androgens and to a lower extent by estrogens (Figure 30) Expression of KLK-L4 in breast cancer tissues and cell lines
  • RNA splicing provides a mechanism whereby protein isoform diversity can be generated and the expression of particular proteins with specialized functions can be restricted to certain cell or tissue types during development (60)
  • the sequence elements in the pre-mRNA at the 5' and 3' splice sites in metazoans have very loose consensus sequence, only the first and the last two bases (GT AG) of the introns are highly conserved (Sambrook, supra)
  • GT AG first and the last two bases
  • the predicted exons of the putative new gene were subjected to homology search using the BLASTN algorithm (37) on the National Center for Biotechnology Information web server (http://www ncbi.nlm.nih.gov/BLAST/) against the human EST database (dbEST).
  • a clone with > 95% homology was obtained from the I.M.A G.E consortium (38) through Research Genetics Inc, Huntsville, AL
  • This clone was propagated, purified and sequenced from both directions with an automated sequencer, using msert- flanking vector primers Rapid amplification of cDNA ends (RACE) According to the EST sequence and the predicted structure of the gene, two sets of gene-specific primers were designed for 5' and 3' RACE reactions.
  • RNA isolated from 26 different human tissues was purchased from Clontech, Palo Alto, CA cDNA was prepared as described below for the tissue culture experiments and used for PCR reactions After aligning all known kalhkrein genes, two primers (KLK-L5-R1 and KLK-L5-F1) (Table 17, SEQ ID NOs 61-64, 9-12, and Figure 32) were designed from areas with relatively low homology Tissue cDNAs were amplified at various dilutions Due to the high degree of homology between kailikreins, and to exclude non-specific amplification, PCR products were cloned and sequenced Normal and malignant breast tissues
  • the breast cancer cell lines BT-474 and T-47D, and the LNCaP prostate cancer cell line were purchased from the American Type Culture Collection (ATCC), Rockville, MD Cells were cultured in RPMI media (Gibco BRL, Gaithersburg, MD) supplemented with glutamme (200 mmol/L), bovme insulin (10 mg/L), fetal bovine serum (10%), antibiotics and antimycotics, in plastic flasks, to near confluency The cells were then ahquoted into 24- well tissue culture plates and cultured to 50% confluency 24 hours before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal- stripped fetal bovine serum For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media at a final concentration of 10 8 M Cells stimulated with 100% ethanol were included as controls The cells were cultured for 24 hours, then harvested for mRNA extraction
  • RNA was extracted from the cell lines or tissues using T ⁇ zol reagent (Gibco BRL) following the manufacturer's instructions RNA concentration was determined spectrophotomet ⁇ cally 2 ⁇ g of total RNA was reverse-transcribed into first strand cDNA using the SuperscriptTM preamplification system (Gibco BRL) The final volume was 20 ⁇ l Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences, two gene-specific primers were designed (KLK-L5-F1 and KLK-L5-R1) (Table 17) and PCR was carried out in a reaction mixture containing 1 ⁇ l of cDNA, 10 M T ⁇ s-HCl (pH 8 3), 50 mM KCl, 1 5 mM MgCl 2 , 200 ⁇ M dNTPs (deoxynucleoside triphosphates), 150 ng of primers and 2 5 units of AmpliTaq Gold DNA poly
  • Signal peptide was predicted using the "SignalP” server (http://www.cbs.dtu.dk/services/ signal) Protein structure analysis was performed by "SAPS” (structural analysis of protein sequence) program (http://dot. ⁇ mgen.bcm.tmc.edu:9331/seq- search struc-predict.html) RESULTS
  • the stop codon is located in the sixth exon, followed by a 3' untranslated region, and a typical polyadenylation signal (AATAAA) is located 16 bp before the poly-A tail ( Figure 33).
  • AATAAA polyadenylation signal
  • This form of KLK-L5 spans a genomic length of 5,801 bp on chromosome 19ql3.3-q l3.4
  • the lengths of the coding regions of the exons are 37, 160, 260, 134, and 156 bp, respectively ( Figures 33 and 34).
  • the predicted protein-coding region is formed of 747 bp, encoding a deduced 248-am ⁇ no acid protein with a predicted molecular mass of 26 7 kDa.
  • the intron/exon splice sites (GT....AG) and their flanking sequences are in agreement with the consensus splice site sequence.
  • the second mRNA form, encoding the KLK-L5-related protein- 1 is an alternatively spliced form in which the last exon is split into two separate exons with an additional intervening intron ( Figure
  • the third mRNA form, encoding for KLK-L5-related prote ⁇ n-2, is similar to the classical form except that the fourth exon is missing ( Figure 32) This leads to frame shifting of the coding region, and an earlier m-frame stop codon will be encountered at position 9,180
  • the protein-coding region of this form consists of 336 bp, encoding toi a pasicted 1 1 1-am ⁇ no acid piotein with a molecular mass of 12 kDa This protein will lack both the serine and aspartate residues characteristic of serine proteases
  • KLK-L5 will possess a trypsin-hke cleavage pattern, similarly to most of the other kailikreins (e g , hKl, hK2, TLSP, neuropsin, zyme, prostase, and EMSP) but different from PSA, which has a serine (S) residue in the corresponding position, and is known to have chymotrypsin like activity (Figure 36) (54) Homology with other members of the kalhkrein multigene family
  • KLK-L5 protein encoded by the KLK-L5 gene is unique, it has a high degree of homology with the other kalhkrein-like genes
  • the KLK-L5 protein (classical form) has 48% amino acid sequence identity and 57% overall similarity with neuropsin, 46% identity with the normal epithelial cell-specific 1 gene product (NESl) and 38% identity with both PSA and hK2 proteins
  • NESl normal epithelial cell-specific 1 gene product
  • a well-conserved peptide motif is found around the amino acid residues of the catalytic triad as is the case with other serine proteases [1 e , histidine (VLTAA /C), serine
  • Figure 36 shows other amino acid residues that are completely conserved between kailikreins and kalhkrein-like proteins
  • Figure 37 shows the phylogenetic analysis which separated the classical kailikreins (hKl, hK2, and PSA) and clustered KLK-L5 with NES 1 and neuropsin proteins in a separate group away from other serine proteases, consistent with previously published studies (27, 41) and indicating that this group of genes probably arose from a common ancestral gene, by gene duplication
  • the KLK-L5 gene is primarily expressed in the salivary gland, stomach, uterus, trachea, prostate, thymus, lung, colon, brain, breast and thyroid gland, but, as is the case with other kailikreins, lower levels of expression are found in some other tissues (testis, pancreas, small intestine, spinal cord).
  • the PCR products were cloned and sequenced The three splice forms of the gene were expressed in most of these tissues However, the relative abundance of each form was different among tissues ( Figure 38).
  • KLK-L5 is under steroid hormone regulation
  • two breast cancer cell lines (BT-474 and T-47D) and a prostate cancer cell line (LNCaP) were used as models.
  • LNCaP cells the gene was up-regulated only by androgen and progestin. Only in this cell line all 3 isoforms were expressed.
  • KLK-L5 was found to be up-regulated, at the mRNA level, by estrogen and androgen, and to a lesser extent by the progestin
  • the rank of potency was estrogen>androgen>progest ⁇ n
  • the rank of potency for the T-47D cell line was androgen>progest ⁇ n>estrogen .
  • the short isoform (related prote ⁇ n-2) was present (Figure 39).
  • KLK-L5 is down regulated in breast cancer
  • KLK-L5 Mapping and chromosomal localization of the KLK-L5 gene
  • PSA and KLK2 transcribe from centromere to telomere; the rest of the genes are transcribed in the reverse direction.
  • the KLKl gene was found to be the most centromeric, and the KLK-L6 gene the most telome ⁇ c (KLK-L6; SEQ.ID. N0.65).
  • KLK-L5 is 21.3 Kb centromeric to KLK- L4 (SEQ.ID.NO. 43) and 1.6 kb more telomeric to the trypsin-hke serine protease gene (TLSP) ( Figure 28)
  • kailikreins are characterized by the following common structural features' (a) All genes are formed of 5 coding exons and 4 intervening introns [some genes may have extra 5( untranslated exon(s)] (14, 35) (b) The exon lengths are usually comparable (c) The intron phases are always conserved (I-II-I-0) (see legend of Figure 34 for definition of intron phases), (d) These genes are clustered in the same chromosomal region, without any intervening non-kal krein-hke genes ( Figure 28) (e) The histidine residue of the catalytic triad of serine proteases is located near the end of the second coding exon; the aspartate residue in the middle of the third coding exon; and serine at the beginning of the fitth coding exon As shown in Figure 34, all these criteria apply to the newly identified KLK-L5 gene Thus, KLK-L5 should be considered a new member of the kalhkre
  • Preproenzymes that contain an N-terminal signal peptide (pre-zymogen), followed by a short activation peptide and the enzymatic domain (41, 54)
  • PreproPSA has 24 additional residues that constitute the pre-region (signal peptide, 17 residues), and the propeptide (7 residues) (67)
  • the signal peptide directs the protein to and across the endoplasmic reticulum (ER) In the ER, the signal peptide is removed and the resulting proPSA is transported to the plasma membrane, where it is secreted
  • the hydrophobicity study ( Figure 35) indicates that the amino terminal region of the KLK-L5 protein is harboring a signal peptide
  • computer analysis of the amino acid sequence of KLK-L5 predicted a cleavage site between ammo acids 17 and 18 (SQA-AT)
  • SQA-AT computer analysis of the amino acid sequence of KLK-L5 predicted a cleavage site between ammo acids 17 and 18
  • KLK-L5 a new member of the human kalhkrein gene family, KLK-L5, has been characterized which maps to the human kalhkrein locus (chromosome 19ql3 3-ql3 4)
  • This gene has two related splice forms in addition to the main form KLK-L5 is expressed in a variety of tissues, appears to be down-regulated in breast cancer and its expression is influenced by steroid hormones Since a few other kailikreins are already used as valuable tumour markers, KLK-L5 may also find a similar clinical application
  • Example 7 Using the Materials and Methods substantially as set out in Example 6, the present inventors identified another novel gene ot the kalhkrein multigen family referred to as KLK-L6
  • KLK-L6 The full structure ot the KLK-L6 gene is shown in Figure 41 Exons 1, 2, 3, 4, 5, 6, and 7 are at nucleic acids 1172-1281, 2561-2695, 2781-2842, 3714-3885, 5715-5968, 6466-6602, and 7258-7520
  • the nucleic acid sequence ot the KLK-L6 gene is also shown in SEQ ID NO 65 and amino acid sequences for the KLK-L6 protein are shown in SEQ ID Nos 66 and 67. (See also GenBank Accession # AF161221)
  • Figure 42 shows a comparative hydrophobicity analysis of KLK-L6 and the prostate-specific antigen (PSA)
  • PSA prostate-specific antigen
  • Figure 44 shows the phylogenetic analysis which separated the classical kailikreins (hKl, hK2, and PSA) and placed KLK-L6 in a separate group
  • (+) >95% homology with published human EST sequences.

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