EP0906423A1 - Metastasis-associated antigen and antibodies thereto - Google Patents

Metastasis-associated antigen and antibodies thereto

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Publication number
EP0906423A1
EP0906423A1 EP97921662A EP97921662A EP0906423A1 EP 0906423 A1 EP0906423 A1 EP 0906423A1 EP 97921662 A EP97921662 A EP 97921662A EP 97921662 A EP97921662 A EP 97921662A EP 0906423 A1 EP0906423 A1 EP 0906423A1
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EP
European Patent Office
Prior art keywords
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arg
leu
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asp
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EP97921662A
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German (de)
English (en)
French (fr)
Inventor
Felix Bachmann
Max M. Burger
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Novartis Pharma GmbH Austria
Novartis AG
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Novartis AG
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Priority claimed from PCT/EP1997/001963 external-priority patent/WO1997041221A1/en
Publication of EP0906423A1 publication Critical patent/EP0906423A1/en
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates to a novel antigen which is closely associated with metastatic behaviour of tumour cells in vitro and in vivo, as well as to antibodies recognising said antigen and uses thereof in diagnosis and treatment of malignancies.
  • Metastasis is a multi-stage process by which tumour cells leave the site of a primary tumour, enter blood and lymph vessels, migrate to distant parts of the body and form novel foci of tumour growth.
  • experimental data suggest that no one factor is exclusively responsible for the determination of metastatic potential. Rather, a cascade of events, each of which incrementally increases the liability of a tumour to metastasis, leads to metastasis and tumour spread.
  • Each of the events of the metastatic cascade is not believed to play a role of identical importance in each metastatic situation.
  • a given metastatic cell is likely to possess some less developed metastatic properties, but still possess high metastatic potential because other properties are highly pronounced.
  • membrane-associated polypeptides having the sequence shown in SEQ ID NOS. 2 and 9, respectively. Also provided are immuno ⁇ genic determinants derived from said polypeptides and antibodies raised thereto. The polypeptides, their derived antigenic determinants and the antibodies are useful for the diagnosis and treatment of metastatic potential in tumours.
  • Figure 1 shows the deduced amino acid sequence of the metastasis-related antigen p150 isolated from mouse. The sequence data derived by amino acid microsequencing is underlined.
  • Figure 2 shows a sequence homology comparison between p150 from mouse and related sequences.
  • an antibody which identifies an antigen preferentially expressed on transformed cells can be identified.
  • This antigen referred to herein as p150 from mouse, has an apparent molecular weight on SDS-PAGE of ca. 150kDa and the sequence shown in SEQ ID No. 2.
  • the human homologue is represented in SEQ ID No. 9.
  • the antigens are expressed in tumours, tumour cell lines and primary cells derived from tumours, and appear at much lower levels in normal human tissue.
  • the levels of expression of the antigens are not consistent between tumours and cell lines derived therefrom, including primary cell lines. However, within each separate system, we have determined that the antigens closely correlate with metastatic potential.
  • the level of expression of the antigens appears to be dependent on the differentiation state of the tumour cell. Thus, relatively undifferentiated tumour cells, which have a high metastatic potential, express relatively high levels of p150, while more differentiated cells express a lower amount of these antigens. Tumour tissue derived from actual metastases appears to express the most elevated levels of p150.
  • the present invention includes the p150 proteins both from mouse and human and all derivatives thereof which are useful for diagnosing the metastatic potential of a tumour.
  • such derivatives are encompassed by the term "p150".
  • the high degree of correlation of p150 with metastatic potential makes the antigens useful for determining the correct treatment to be used, as it is known that a high metastatic potential correlates with a poor prognosis.
  • the derivatives of p150 comprised in the invention include all those which retain characteristics of p150 which allow them to be identified in diagnostic assays.
  • the preferred characteristic retained by the p150 derivatives of the invention is a common antigenic determinant shared with p150.
  • “Common antigenic determinant” means that the derivative in question at least one antigenic function of p150.
  • Antigenic functions includes possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured p150 polypeptide or fragment thereof.
  • p150 as provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants, glycosylation variants and other covalent derivatives of p150 which retain the physiological and/or physical properties of p150.
  • Exemplary derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid.
  • Such a moiety may be a detectable moiety such as an enzyme or a radioisotope.
  • a detectable moiety such as an enzyme or a radioisotope.
  • naturally occurring variants of p150 found with a particular species preferably a mammal.
  • Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of the p150 gene.
  • Derivatives which retain common antigenic determinants can be fragments of p150.
  • Fragments of p150 comprise individual domains thereof, as well as smaller polypeptides derived from the domains.
  • smaller polypeptides derived from p150 according to the invention define a single epitope which is characteristic of p150. Fragments may in theory be almost any size, as long as they retain one characteristic of p150. Preferably, fragments will be between 5 and 500 amino acids in length. Longer fragments are regarded as truncations of the full-length p150 and generally encompassed by the term "p150".
  • Derivatives of p150 also comprise mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain at least one feature characteristic of p150.
  • conservative amino acid substitutions may be made substantially without altering the nature of p150, as may truncations from the 5' or 3' ends.
  • Deletions and substitutions may moreover be made to the fragments of p150 comprised by the invention.
  • p150 mutants may be produced from a DNA encoding p150 which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids.
  • substitutional, deletional or insertional variants of p150 can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of p150.
  • the fragments, mutants and other derivative of p150 preferably retain substantial homology with p150.
  • homology means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function.
  • homology is used to refer to sequence identity.
  • the derivatives of p150 preferably retain substantial sequence identity with the sequences of SEQ ID Nos. 2 and 9, respectively.
  • Substantial homology where homology indicates sequence identity, means more than 50% sequence identity, preferably more than 75% sequence identity and most preferably a sequence identity of 90% or more.
  • the proteins or derivatives thereof of the invention are provided in isolated form.
  • isolated means that the respective protein or derivative thereof has been identified and is free of one or more components of its natural environment.
  • Isolated pi 50 includes p150 in a recombinant cell culture. P150 present in an organism expressing a recombinant p150 gene, whether the p150 protein is "isolated” or otherwise, is included within the scope of the present invention.
  • the polypeptides according to the invention are closely associated with the metastatic process in tumour cells. Accordingly, the invention provides a composition comprising a polypeptide according to the invention or an antagonist thereto for use as a medicament in the treatment or diagnosis of tumours.
  • nucleic acids encoding p150 there are provided nucleic acids encoding p150.
  • these nucleic acids are also useful as probes, thus readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding p150.
  • the nucleic acid may be unlabelled or labelled with a detectable moiety.
  • nucleic acids according to the invention are useful e.g. in a method determining the presence of p150-specific nucleic acid, said method comprising hybridising the DNA (or RNA) encoding (or complementary to) p150 to test sample nucleic acid and determining the presence of p150.
  • the invention provides nucleic acid sequences that are complementary to, or hybridise under stringent conditions to, a nucleic acid sequence encoding p150.
  • the invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) p150.
  • the nucleic acid is DNA and further comprises a replicable vector comprising the nucleic acid encoding p150 operably linked to control sequences recognised by a host transformed by the vector.
  • the invention provides host cells transformed with such a vector and a method of using a nucleic acid encoding p150 to effect the production of p150, comprising expressing p150 nucleic acid in a culture of the transformed host cells and, if desired, recovering p150 from the host cell culture.
  • the present invention relates to isolated p150 proteins and derivatives thereof encoded by the above-described nucleic acids.
  • Isolated p150 nucleic acid includes a nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of p150 nucleic acid or in crude nucleic acid preparations, such as DNA libraries and the like. Isolated nucleic acid thus is present in other than in the form or setting in which it is found in nature.
  • isolated p150 encoding nucleic acid includes p150 nucleic acid in ordinarily p150- expressing cells where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature.
  • isolated nucleic acids e.g. DNAs or RNAs, encoding p150, particularly mammalian p150, e.g. human p150, or fragments thereof.
  • the invention provides DNA molecules encoding p150, or a fragment thereof.
  • a DNA comprises a coding single stranded DNA, a double stranded DNA of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself.
  • Exemplary nucleic acids encoding p150 are represented in SEQ ID Nos. 1 and 8.
  • the preferred sequences encoding p150 are those having substantially the same nucleotide sequence as the coding sequences in SEQ ID Nos. 1 and 8, with the nucleic acids having the same sequence as the coding sequences in SEQ ID Nos. 1 and 8 being most preferred.
  • nucleotide sequences which are substantially the same share at least about 90 % identity. However, in the case of splice variants having e.g. an additional exon sequence homology may be lower.
  • nucleic acids of the invention are preferably substantially homologous to the sequences of p150 as shown in SEQ ID Nos. 1 and 8.
  • the terms "substantially” and “homologous” are used as hereinbefore defined with reference to the p150 polypeptide(s).
  • nucleic acids according to the invention are fragments of the p150-encoding sequences, or derivatives thereof as hereinbefore defined in relation to polypeptides. Fragments of the nucleic acid sequences of a few nucleotides in length, preferably 5 to 150 nucleotides in length, are especially useful as probes.
  • Exemplary nucleic acids can alternatively be characterised as those nucleotide sequences which encode a p150 protein and hybridise to the DNA sequences set forth SEQ ID Nos. 1 and 8, respectively, or a selected fragment of said DNA sequences. Preferred are such sequences encoding p150 which hybridise under high-stringency conditions to the sequences of SEQ ID Nos. 1 and 8, respectively.
  • Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T m ) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
  • high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na + at 65-68 °C.
  • High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na + pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor.
  • high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - 0.1x SSC, 0.1 % SDS.
  • Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridisation temperature in 1x SSC, 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0.1 % SDS.
  • nucleic acids of the invention are obtainable according to methods well known in the art.
  • a DNA of the invention is obtainable by chemical synthesis, using polymerase chain reaction (PCR) or by screening a genomic library or a suitable cDNA library prepared from a source believed to possess p150 and to express it at a detectable level.
  • PCR polymerase chain reaction
  • Chemical methods for synthesis of a nucleic acid of interest include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports. These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.
  • cDNA expression libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it.
  • suitable means include monoclonal or polyclonal antibodies that recognise and specifically bind to p150; oligonucleotides of about 20 to 80 bases in length that encode known or suspected p150 cDNA from the same or different species; and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridising gene.
  • Appropriate probes for screening genomic DNA libraries include, but are not limited to oligonucleotides, cDNAs or fragments thereof that encode the same or hybridising DNA; and/or homologous genomic DNAs or fragments thereof.
  • a nucleic acid encoding p150 may be isolated by screening suitable cDNA or genomic libraries under suitable hybridisation conditions with a probe, i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID Nos. 1 and 8.
  • a probe i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID Nos. 1 and 8.
  • Suitable libraries are commercially available or can be prepared e.g. from cell lines, tissue samples, and the like.
  • a probe is e.g.
  • nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised.
  • the nucleotide sequences are usually based on conserved or highly homologous nucleotide sequences or regions of p150.
  • the nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known.
  • nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation.
  • suitable label means is a radiolabel.
  • the preferred method of labelling a DNA fragment is by incorporating a 32P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.
  • Oligonucleotides are usually end-labelled with g 32P -labelled ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • positive clones are identified by detecting a hybridisation signal; the identified clones are characterised by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding a complete p150 (i.e., if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones.
  • the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNAs and deduced amino acid sequences provided herein.
  • nucleotide sequences of the invention may be used as hybridisation probes.
  • Such probes can e.g. be used to screen specific tissues and body fluids like e.g. blood, urine, spinal- fluid, ascites and serum.
  • antisense-type therapeutic agents may be designed.
  • nucleic acids of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof.
  • Such mutants can be used e.g. to produce a p150 mutant that has an amino acid sequence differing from the p150 sequences as found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridise to produce secondary mRNA structure such as loops or hairpins.
  • vector refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible.
  • the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
  • Both expression and cloning vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, these sequences are those which enable the vector to replicate independently of the host chromosomal DNA, and include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.
  • Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression.
  • a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome.
  • DNA may also be replicated by insertion into the host genome.
  • the recovery of genomic DNA encoding p150 is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise p150 DNA.
  • DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.
  • an expression and cloning vector may contain a selection gene also referred to as selectable marker.
  • This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
  • any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.
  • Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3. LEU2, LYS2. TRP1. or HIS3 gene. Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from ] ⁇ . coli plasmids, such as pBR322, Bluescript ® vector or a pUC plasmid, e.g. pUCl ⁇ or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up p150 nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase or genes conferring resistance to G418 or hygromycin.
  • the mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive.
  • selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes p150.
  • Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesised from thus amplified DNA.
  • Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to p150 nucleic acid. Such a promoter may be inducible or constitutive.
  • the promoters are operably linked to DNA encoding p150 by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native p150 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of p150 DNA.
  • Promoters suitable for use with prokaryotic hosts include, for example, the b-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding p150, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding p150.
  • the p150 genes according to the invention preferably include a secretion sequence in order to facilitate secretion of the corresponding polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
  • the peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.
  • Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene.
  • GAP glyceraldehyde-3-phosphate dehydrogenase
  • PGK 3-phospho glycerate kinase
  • TATA binding protein (TBP) gene can be used.
  • hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene
  • a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene PH05-GAP hybrid promoter
  • a suitable constitutive PHO5 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.
  • p150 gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with p150 sequence, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or
  • Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to p150 DNA, but is preferably located at a site 5' from the promoter.
  • a eukaryotic expression vector encoding p150 may comprise a locus control region (LCR).
  • LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the p150 gene is to be expressed in the context of a permanently- transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, in vectors designed for gene therapy applications or in transgenic animals.
  • Suitable eukaryotic host cells for expression of p150 include yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms will also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNAs encoding p150.
  • An expression vector includes any vector capable of expressing p150 nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • DNAs encoding p150 may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).
  • a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).
  • Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of p150.
  • transient expression systems are useful e.g. for identifying p150 mutants, to identify potential phosphorylation sites, or to characterise functional domains of the protein.
  • Plasmids employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing p150 expression and function are known to those skilled in the art.
  • Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
  • cells containing the above-described nucleic acids there are provided cells containing the above-described nucleic acids.
  • host cells such as prokaryote, yeast and higher eukaryote cells may be used for replicating DNA and producing p150.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5a and HB101 , or Bacilli.
  • Further hosts suitable for p150 encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae.
  • Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells.
  • vertebrate cells in recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells.
  • the host cells referred to in this disclosure comprise cells in in vitro culture as well as cells that are within a host animal.
  • DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art.
  • Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.
  • the cells should be transfected with a sufficient amount of p150-encoding nucleic acid to form p150.
  • the precise amounts of DNA encoding p150 may be empirically determined and optimised for a particular cell and assay.
  • Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.
  • Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby p150 encoded by the DNA is expressed.
  • the composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.
  • DNAs provided herein may be expressed in any suitable host cell, e.g. those referred to above, preferred for expression of DNA encoding functional p150 are eukaryotic expression systems such as baculovirus-based systems and, particularly, mammalian expression systems, including commercially available systems and other systems known to those of skill in the art.
  • eukaryotic expression systems such as baculovirus-based systems and, particularly, mammalian expression systems, including commercially available systems and other systems known to those of skill in the art.
  • p150 encoding DNA is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express p150.
  • the resulting cell lines can then be produced in quantity for reproducible qualitative and/or quantitative analysis of the effect(s) of potential drugs affecting p150 function.
  • p150 expressing cells may be employed for the identification of compounds, particularly small antagonising molecules interfering with p150 function are useful.
  • An alternative to achieve an antagonistic effect is to rely on over-expression of antisense p150 RNA.
  • host cells expressing p150 are useful for drug screening and it is a further object of the present invention to provide a method for identifying compounds which modulate and preferable reduce the activity of p150, said method comprising exposing cells containing heterologous DNA encoding p150, wherein said cells produce functional p150, to at least one compound or signal whose ability to modulate the activity of said p150 is sought to be determined, and thereafter monitoring said cells for changes caused by said modulation.
  • Such an assay enables the identification of agonists, antagonists and allosteric modulators of p150.
  • Cell-based screening assays can be designed e.g. by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, such as b galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on p150.
  • a reporter protein i.e. an easily assayable protein, such as b galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • luciferase e.g. an easily assayable protein, such as b galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase.
  • CAT chloramphenicol acetyltransfer
  • the present invention also provides a method to exogenously affect p150 dependent processes occurring in such cells.
  • Recombinant p150 producing host cells e.g. mammalian cells
  • the modulating effect(s) thereof can then be evaluated by comparing the p150-mediated response in the presence and absence of test compound, or relating the p150-mediated response of test cells, or control cells (i.e., cells that do not express p150), to the presence of the compound.
  • a compound or signal that modulates the activity of p150 refers to a compound that alters the activity of p150 in such a way that the activity of p150 is different in the presence of the compound or signal (as compared to the absence of said compound or signal).
  • the invention also provides a transgenic non-human mammal which has been modified to modulate the expression of endogenous p150.
  • the transgenic non-human mammal is a transgenic mouse.
  • a transgenic mouse may be designed in which p150 production is greatly reduced or eliminated
  • the transgenic mouse of the invention may express elevated levels of p150, or may be subject to regulation of p150 expression in a developmentally or tissue-specific manner, or via control by exogenous agents. Study of such an animal provides insights into the importance of p150 in vivo.
  • the invention provides a composition comprising a nucleic acid encoding a polypeptide according to the invention or an antagonist thereto for use as a medicament in the treatment or diagnosis of tumours.
  • a transcription unit encoding a polypeptide according to the invention or an antagonist thereto for use in a method of treatment of a tumour or other condition, for example a condition involving aberrant p150 gene expression, by gene therapy techniques.
  • the transcription unit provided according to the present aspect of the invention comprises regulatable control regions which include a promoter, together with one or more enhancers and/or LCRs.
  • the transcription unit may be delivered to the subject by any suitable means, including viral vectors, especially retroviral vectors, adeno- and adeno associated viral vectors, non-viral delivery systems, including liposomal and antibody targeted delivery systems, and direct uptake of naked DNA.
  • the target tissue is advantageously a tumour tissue.
  • antibodies specifically recognising and binding to p150 may be generated against the p150 having the amino acid sequences set forth in SEQ ID Nos. 2 and 9, respectively.
  • p150 or p150 fragments (which may also be synthesised by in vitro methods) are fused (by recombinant expression or an in vitro peptidyl bond) to an immunogenic polypeptide and this fusion polypeptide, in turn, is used to raise antibodies against a p150 epitope.
  • Anti-p150 antibodies are recovered from the serum of immunised animals. Alternatively, monoclonal antibodies are prepared from cells in vitro or from in vivo immunised animals in conventional manner. Preferred antibodies identified by routine screening specifically identify p150 on tumour cells with a high metastatic potential.
  • the antibodies of the invention are useful for studying p150 tissue localisation, screening of an expression library to identify nucleic acids encoding p150 or the structure of functional domains, as well as for the purification of p150, and the like. Preferably, however, they are useful in the diagnosis and treatment of tumours and especially tumour metastasis.
  • Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover, the invention includes antibody fragments, such as Fab, F(ab') 2 , Fv and ScFv. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
  • the antibodies according to the invention are especially indicated for the diagnosis and therapy of cancer. Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in a tumour in vivo. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient. Moreover, the may be fluorescent labels or other labels which are visualisable on tissue samples removed from patients.
  • chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications.
  • immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [see international patent application WO 90/07861 (Protein Design Labs)].
  • Antibodies according to the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture.
  • monoclonal antibodies according to the invention are antibody 6G7 (DSM 2256) and antibody 6G10 (DSM 2257).
  • Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferable in mammalian cell culture.
  • the selected cell culture system preferably secretes the antibody product.
  • the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.
  • a host e.g. E. coli or a mammalian cell
  • a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.
  • Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPM1 1640 medium, optionally replenished by a mammalian serum, e.g. fetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like.
  • suitable culture media which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPM1 1640 medium
  • a mammalian serum e.g. fetal calf serum
  • trace elements and growth sustaining supplements e.g. feeder cells
  • feeder cells such as normal mouse peritoneal exudate cells, sple
  • Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, N2CYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies.
  • Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
  • the desired antibodies can also be obtained by multiplying mammalian cells in vivo.
  • hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours.
  • the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection.
  • pristane tetramethyl-pentadecane
  • hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
  • the cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing p150, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • an enzyme immunoassay e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like.
  • the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno)-affinity chromatography, e.g. affinity chromatography with p150 protein or with Protein-A.
  • the invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention.
  • the preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.
  • the invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to p150, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with purified p150 protein, an antigenic carrier containing purified p150 or with cells bearing p150, antibody-producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected.
  • a suitable mammal for example a Balb/c mouse
  • spleen cells of Balb/c mice immunised with cells bearing p150 are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.
  • a fusion promoter preferably polyethylene glycol.
  • the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000.
  • the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
  • the invention also concerns recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to the extracellular domain of p150 as described hereinbefore.
  • DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.
  • DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed p150 can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.
  • a mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody.
  • Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s).
  • Such a mutant sequence is also a degenerated sequence.
  • Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.
  • mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
  • the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.
  • the invention therefore also concerns recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed p150 fused to a human constant domain g, for example g1 , g2, g3 or g4, preferably g1 or g4.
  • the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to p150 fused to a human constant domain k or I, preferably k.
  • the invention pertains to recombinant DNAs coding for a recombinant antibody wherein the heavy chain variable domain and the light chain variable domain are linked by way of a DNA insert coding for a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a DNA coding for a cleavage site and/or a DNA coding for a peptide spacer and/or a DNA coding for an effector molecule.
  • the DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications.
  • effector molecules which are toxins or enzymes, especially enzymes capable of catalysing the activation of prodrugs, are particularly indicated.
  • the DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.
  • Antibodies and antibody fragments according to the invention are useful in the diagnosis and therapy of tumours. Accordingly, the invention provides a composition for the therapy or diagnosis of tumours comprising an antibody according to the invention.
  • the antibody is preferably provided together with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotopic or other means. These means can e.g. be used to screen specific tissues and body fluids like e.g. blood, serum, ascites, spinal-fluid and urine.
  • the antibody and the detection means may be provided for simultaneous, simultaneous separate or sequential use, in a diagnostic kit intended for the diagnosis of tumour conditions.
  • the invention further provides a method for determining the metastatic potential of a tumour cell, comprising assessing the level of p150 expression in said cell, a higher level of p150 expression being indicative of a lower state of differentiation and consequently a higher metastatic potential.
  • the invention provides a method for the therapy of tumours comprising administering to a patient a therapeutically effective dose of an antibody, a polypeptide or a nucleic acid according to the invention.
  • a therapeutically effective dose of an antibody, a polypeptide or a nucleic acid according to the invention comprising administering to a patient a therapeutically effective dose of an antibody, a polypeptide or a nucleic acid according to the invention.
  • Membrane-associated proteins are isolated from human placental tissue. The rationale for the selection of placental tissue is that since the trophoblast invades surrounding tissue, it is likely that invasion-associated proteins will be expressed therein.
  • 200 g fresh human placenta is used as a starting material.
  • the placenta is cut into small pieces and then washed several times in ice cold PBS in order to remove blood.
  • the small pieces are transferred into a beaker containing 200 ml lysis buffer (100 mM Octyl-beta-D- glucoside, 1 mM PMSF, 5 mg/ ml Leupeptin and 1 mM Mn 2+ dissolved in PBS ), homogenised with a Warren blender, stirred for 4 h at 4 °C and then once again homogenised with a Polytron. Solid matter is then removed by centrifugation at 25000g for 60 minutes.
  • Balb/c mice are then used for raising Mabs against putative RGD-binding proteins. Each animal receives about 50-100 mg of partially purified membrane proteins derived from the Sepharose ® -GRGDSP column, plus 0.5 ml Freunds Adjuvants injected ip. As booster applications, the animals twice receive 50-100 mg of protein plus 0.5 ml Freunds incomplete Adjuvants every 3-4 weeks. Finally, 2 days before the fusion of the splenocytes with the myeloma cells ( PAI cells), the animals receive another 20-30 mg of protein intravenously. The resulting Mabs are screened for cell surface staining and expression levels of the newly defined antigens in transformed versus non transformed cells. Comparison is made between 3T3 cells in a non-transformed state, and 3T3 cells transformed with src or ras, as well as CEF68 cells grown at permissive or non-permissive temperatures.
  • Cell surface staining is analysed by immunofluorescence, as follows. Cells are grown on glass coverslips or on glass slides, washed in PBS or in DME and fixed for 30 min. in formaldehyde 3.7% in PBS or in DME at room temperature (20°C). Cells are then permeabilised in 0.5 % TX-100 ( in PBS or DME ) for 1-3 min at room temperature. Subsequently, cells are washed 3-4 times in PBS or DME at room temperature. Liquid is drained off and 0.03ml of a first antibody raised against the membrane-associated protein fractions diluted in DME / 10% FCS is added. The cells are incubated for 60 minutes at 37°C in a humid incubator.
  • the cells are washed 3-4 times in PBS or DME at room temperature. 0.03 ml of a second, anti-mouse antibody labelled with FITC or TRITC diluted in DME / 10% FCS is added, and the cells incubated for 60 minutes at 37°C in a humid incubator. Subsequently, they are washed 3-4 times in PBS or DME and once in water. They are then embedded in an appropriate mounting medium and stored in the dark. Fluorescence can then be assessed by viewing the embedded cells using suitable fluorescence microscopy equipment.
  • melanoma cells are incubated with the MAb prior to tail vein injection into mice. After 3 weeks the animals are sacrificed and the lung colonies counted. A 80-85% inhibition of the lung colonisation ability can be observed.
  • treatment number of lung colonies mean std %inhibition control 25,16,2,1,14,20,25,24 16 10 H11VII 1,0,5,9,0,4,0,0,5,9 3 4 80
  • treatment number of lung colonies mean std %inhibition untreated 36,45,23,20,52,64,>300(64), 46
  • treatment number of lung colonies mean std fold indue. control 2,6,5,5,13,0,3,1,2,35 4 4
  • a general problem by using antibodies in an in vivo system is the possible stimulation of the host immune system. Especially the cytotoxic as well as the natural killer activities towards cells tagged with antibodies can be influenced. Due to the fact that peripheral cellular blood components do not express p150 in detectable amounts, it is possible to reduce the influence of the host immune system by changing the treatment. Instead of incubation of the cancer cells with Mabs prior to injection we pretreat the host animals 24 hrs before they received F1 cells. The results of this experiment, in which the original MAb (H11 VII) as well as a new MAb raised against mouse p150 (6G7) are used, are outlined in Table 2.
  • pretreatment number of lung colonies mean std %inhibition control 1 ,4,13,31 ,40,44,63,>99(63),>99(63),>99(63 39 25 n/a
  • Mice are pretreated with the monoclonal antibodies for 24hours before injection of B16 cells. O.5mg of antibody is injected i.p.
  • Antibodies are prepared by precipitation in 28g/100ml (NH 4 ) 2 S0 4 and ConA chromatography.
  • antibody H11 VII is seen to recognise bands on SDS-PAGE migrating at approximately 28, 35, 70 and 150 KDa.
  • the bands are purified from the gel and rabbit polyclonal antisera raised thereto.
  • the antiserum raised against the 28KDa band cross-reacts with the 150KDa band on western blots.
  • the antigen recognised by MAb H1 1VII can be identified as a 150 kDa membrane associated protein (p150).
  • P150 is expressed in B16F1 cells in large amounts. Therefore, these cells are used as starting material for the purification of p150.
  • a crude membrane fraction is isolated from B16F1 cells by centrifugation at 100,000g and lysis in 1%NP40, 2mM Tris-HCl pH 7.5, containing PMSF and aprotinin protease inhibitors. The supernatant is loaded on a Sepharose ® Q column, run off, transferred to a Sepharose ® S column, again run off, and separated by reducing SDS-PAGE on a 5% gel. The 150KDa band is electrocuted.
  • P150 is microsequenced by digestion with trypsin, V8 and Lys-C followed by sequencing using Edman degradation on an Applied Biosystems sequencer. An oligonucleotide deduced from the sequence used to probe a mouse melanoma B16F1 library.
  • centrosomin A 280 amino acids from the sequence shown in SEQ ID No. 3
  • B 430 amino acids from the sequence shown in SEQ ID No. 4
  • centrosomin A 280 amino acids from the sequence shown in SEQ ID No. 3
  • centrosomin B 430 amino acids from the sequence shown in SEQ ID No. 4
  • a yeast ORF (YBR079; SEQ ID No. 5) showing 51% similarity and 29% identity, a tobacco ORF (NTPNL35; SEQ ID No. 6) with 59% similarity and 37% identity and an ORF found in nematode C.elegans (EGL-45; SEQ ID No. 7) showing 56% similarity and 36% identity.
  • EGL-45 the two other ORFs are of unknown function. Mutational studies performed on EGL-45 suggest an involvement of this protein in the regulatory steps of the hermaphrodite specific neuronal differentiation. Expression of EGL-45 is found in most cells of the worm. Null mutants give rise to a sterile or even a lethal phenotype.
  • antibody (dilution 1/10 - 1/100) is added to adherent cells or detached cells (EDTA, PBS w/o Ca 2+ / Mg 2+ , wash once in PBS /1% BSA, spin down and add the antibody).
  • Cells are incubated either on the tissue culture plate ( at 20 or 37°C ) or in an Eppendorf tube for 1-2 h. Subsequently, cells are washed with PBS /1% BSA on the plate and detached, or washed by spinning down in the Eppendorf tube and adding PBS /1 % BSA. The washing step is repeated 3-4 times.
  • a second antibody conjugated with FITC or TRITC is added to the cells in PBS /1% BSA and incubated for 1 h at 20°C while slowly shaking. The cells are washed 2-3 times with PBS /1% BSA. A FACscan may then be carried out according to standard protocols.
  • FACS analysis demonstrates that P150 is clearly expressed at the cell surface of B16 F1 mouse melanoma cells.
  • the labelling pattern as well as the intensity of the staining is independent of the fact whether the cells were labelled in suspension or they were attached to tissue culture dishes.
  • a confluent 15 cm dish of B16 F1 or HeLa cells is washed 3-4 times with cold PBS.
  • 10 ml PBS containing 0.05 ml Biotinamidocaproate-N- Hydroxysuccimide Ester (Sigma B 2643), which was dissolved in DMSO at 100mg/ml, is then added and the cells incubated for 40 - 60 minutes in the cold room (4°C).
  • the cells are then washed 2-3 times with cold PBS and lysed in cold lysis buffer (see p150 purification) for 30 minutes on ice. Undissolved material is spun down at 13000g for 10 minutes and the cleared cell lysate incubated with Avidin Agarose beads for 30 minutes on a shaker in the cold room.
  • the Avidin Agarose beads are washed with lysis buffer 3-4 times, once with cold ddH 2 0 and boiled in SDS-PAGE sample buffer. SDS-PAGE is run and western blotting carried out according to standard protocols.
  • B16 F1 cells are seen to express p150 on the cell surface as determined by the above cell surface biotinylation method. However only a very small proportion of total cellular p150 is exposed at the cell surface. This seems to be involved in cell-cell and/or in cell- extracellular matrix interactions as already indicated by the results obtained from the in vivo colonisation experiments. Contrary to this finding in B16 cells, in HeLa cells no cell surface biotinylation of p150 could be observed.
  • p150 contains a hydrophobic stretch of 70 aa (1050- 1120), which could serve as a transmembrane domain. However, most of the cellular p150 is found in the cytoplasmic compartment associated with endogenous membranes.
  • p150 On HeLa cells, in contrary to the B16F1 cells, p150 could not be detected on the cell surface by biotinylation technique. Therefore, it might be possible, that the distribution of p150 depends on the cell type and on its status.
  • B16 cells show a strong transformed phenotype; e.g. they do not spread out very well in cell culture. As a consequence, the cytoskeletal elements are not as well separated as in cells which show a more spread out phenotype. Therefore we chose HeLa cells for our localisation studies. Using confocal microscopy, a strong perinuclear staining is observed, while throughout the cytoplasm staining is associated with vescicular structures. In cells undergoing mitosis, staining is observed to be associated with the midbody, in a pattern similar to that of tubulin.
  • p150 is normally expressed in several mouse organs. We find a high expression level in pancreas, as well as liver, although signal in liver might be artefact due to high protein concentration in that region, and a medium level in brain, thymus, and testis. Low expression of p150 occurs in lung, spleen and kidney. P150 expression in testis could reflect the fact that there we can find a lot of undifferentiated, proliferating cells. In testis, p150 and b-tubulin codistribute on structures resembling sperm tails.
  • HBL 100, BT 20, T47D, SKBR3, MB231 and MB453 Some breast cancer cell lines (HBL 100, BT 20, T47D, SKBR3, MB231 and MB453), a liver cancer cell line (Hep G2) as well as a ovarian cancer cell line (SKO V3) are investigated.
  • the p150 levels are different in these lines, but p150 appears to be universally present.
  • cancer tissue and non-cancerous normal tissue is chosen macroscopically by an experienced pathologist for transfer to the laboratory.
  • the tissue arrives in the laboratory about 5 hours after mastectomy at latest, is frozen and stored at -80°C.
  • Corresponding cancer and normal tissue are fixed in 10% buffered formalin and embedded in paraffin.
  • 5 micron sections are cut for hematoxylin/eosin staining.
  • For immunohistochemistry the 5 micron sections are mounted on cementit (Merz und Benteli
  • Frozen human tumor and normal tissue is cut into small pieces, suspended in STE buffer (0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 1 mM EDTA) with protease inhibitors aprotinin 2 ⁇ g/ml, leupeptin 1 ⁇ g/ml and 1 mM PMSF and homogenized with a Polytron homogenizer.
  • the homogenates are centrifuged at 1000 g for 15 minutes and the supernatants are mixed with one volume of 2x sample buffer consisting of 0.1 M Tris-HCl pH 6.8, 4% SDS and 20 % glycerol. This mixture is incubated for 30 minutes at 56°C and recentrifuged at 15,000 g for 10 minutes.
  • the protein concentration of the supernatants is determined with a BCA protein assay kit (Pierce, Rockford, IL).
  • Extracts of cells or surgical specimens containing 20 or 40 ⁇ g of protein are subjected to electrophoresis in the presence of 0.1% SDS in 5% SDS-PAGE minigels. After electrophoresis, the proteins are transferred electrophoretically at 1 mA per cm 2 for 90 minutes onto a PVDF membrane.
  • the membranes are blocked in blocking solution (0.1 M Tris-HCl pH 7.4, 0.1 M MgCI 2 , 0.5% Tween 20, 1% Triton X-100, 1 % BSA, 5% FCS) for 30 minutes at room temperature (RT), decorated with a chicken Ab against p150 (Sophie 1 :2000) or with Mab 6G10 (supernatant 1 :20) overnight at 4°C, washed with blocking solution and then incubated for 1 hour at RT with a goat anti-chicken peroxidase-coupled second antibody (1 :2000, Southern Biotechnology Ass.
  • blocking solution 0.1 M Tris-HCl pH 7.4, 0.1 M MgCI 2 , 0.5% Tween 20, 1% Triton X-100, 1 % BSA, 5% FCS
  • tissue blocks of normal and carcinoma tissue are taken from three cases for immunohistological examination.
  • the 5 micron paraffin sections are deparaffinized, rehydrated and washed twice in Tris-NaCI buffer (30 mM Tris (pH 7.5), 0.9% NaCl). Internal peroxidases are blocked with blocking solution (25 mM ⁇ -D-glucose, 25 mM sodium azide and 0.6 units/ml glucose oxidase (Sigma, Switzerland) in Tris-NaCI buffer) for 1 hour at 37°C. After washes in Tris-NaCI buffer, the sections are heated in 500 ml citrate buffer (0.1 mM, pH 6.0) in the microwave oven operated at 750 W for 10 minutes.
  • the sections are allowed to cool down and washed again in Tris-NaCI buffer before blocking with 1 % BSA in Tris-NaCI buffer for 15 minutes.
  • the chicken antibody against p150 Sophie 1/1000 is applied for 60 minutes at RT in the same buffer, whereas the Mab 6G7, the monospecific rabbit anti- NH 2 terminal peptide (225 amino acids) antibody and the Mab against Ki-67 (MIB1 , Dianova GmbH, Germany) are incubated with the sections overnight at 6°C. Subsequently the sections are washed twice, incubated with second antibodies (goat anti- chicken, goat anti-mouse or goat anti-rabbit; all peroxidase coupled, diluted 1 :2000 , Southern Biotechnology Ass.
  • the mean age of our 22 patients with primary breast cancer when operated was 64 years (range 37-90 y) and the mean tumor size was 48 mm (range 20-140 mm). 55% of the tumors were poorly differentiated (Gill according to the Elston and Ellis grading system (12)) while 14% were well differentiated. The predominant histological type (91%) was ductal invasive, the others were lobular invasive tumors. Metastasis to axillary lymph nodes was detected in 59%, in 27% the lymph nodes showed no metastasis and from all other patients no lymph nodes were available. In 21 of the 22 patients Western blotting have revealed significantly higher p150 expression in tumors than in the normal tissue counterparts of the same patients.
  • a distinct pattern of p150 expression is observed in breast cancer tissue versus the control tissue. Invasive and in situ ductal cancer react equally positive. Necrotic tissue is found to be negative for p150 expression. Normal non proliferating tissue shows faint focal cytoplasmic staining of luminal epithelial cells as do myoepithelial cells. A few nuclei in proliferating epithelial cells also present positive staining. Stromal tissue is negative for p150 in all cases.
  • Proliferative and precancerous alterations in breast tissue such as epitheliosis, apocrine metaplasia and adenosis show staining for p150. No correlation between Ki-67 staining and p150 expression in normal breast tissue is observed.
  • the mean age of the 31 patients with primary breast cancer when operated was 61 years (range 36-85 years) and the mean tumor size was 28 mm (range 9-90 mm). Fifty-five percent of the cancers were poorly differentiated (Gill according to the Elston and Ellis grading system), 32% were moderately differentiated and 13% were benign. The predominant histological type (67%) was ductal invasive, the others were lobular invasive (16%), fibroadenomas (10%), one squamous cell carcinoma, one a mucinous carcinoma and one was a papilloma.
  • lymphnode Metastasis to lymphnode was detected in 42%, in 29% no lymph nodes were involved and in 29% of the patients lymph nodes were not available. Thirty of 31 patients show significantly higher p150 expression in tumors than in normal tissue counterparts in Western blot analysis.
  • p150 In contrast to mammary carcinoma, malignancy in cervical squamous cell carcinoma inversely correlates with p150 expression levels. Moreover, the behavior of neoplastic tissue in terms of p150 expression strongly depends on the histological origin. Therefore, it is not a general issue for p150, that expression levels increase with enhanced proliferative ability. Thus it appears that p150 can be used both as a dedifferentiation and as a proliferation marker.
  • Antibodies according to the invention have been deposited in accordance with the
  • GCC AAC GAA TTT CTT GAG GTT GGC AAA AAG CAG CCT GCC CTG GAT GTT 217 Ala Asn Glu Phe Leu Glu Val Gly Lys Lys Gin Pro Ala Leu Asp Val 15 20 25 30
  • AAG AGC CAC TTG GCT AAG GAA GGG TTA TAT CAA TAT AAG AAC ATA TGC 361 Lys Ser His Leu Ala Lys Glu Gly Leu Tyr Gin Tyr Lys Asn lie Cys 65 70 75
  • ATC AAG AAG AAA ACT GTT CGG GAG CGG 2041 lie Leu Gin Glu His Glu Gin lie Lys Lys Lys Thr Val Arg Glu Arg 625 630 635
  • ATT GAC ATT GAA GAC CTT GAA GAA CTG GAT CCA GAT TTT ATT ATG GCC 2137 lie Asp lie Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe lie Met Ala 655 660 665 670 AAA CAG GTT GAA CAA CTG GAG AAA GAA AAG AAG GAA CTT CAG GAA CGC 2185 Lys Gin Val Glu Gin Leu Glu Lys Glu Lys Lys Glu Leu Gin Glu Arg 675 680 685
  • GTC AAG AAA CTA GAA GAA GTA GAA AGG AAA AAG CGG CAA AGA GAG CTG 2713 Val Lys Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gin Arg Glu Leu 850 855 860
  • Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn lie Gin Arg Glu Lys Glu 580 585 590 Glu Leu Glu Gin Arg Glu Ala Glu Leu Gin Lys Tyr Glu Arg Leu Lys 595 600 605
  • Arg Lys lie Thr Tyr Tyr Arg Glu Arg Lys Lys Lys Ser Arg Gly Gly 805 810 815
  • CTAGGTCTTC AAGCCCCACC GACACGAATT GGCCTTATTA ATGATATGGT CAGATTTAAT 1260

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EP97921662A 1996-04-29 1997-04-18 Metastasis-associated antigen and antibodies thereto Withdrawn EP0906423A1 (en)

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US1648796P 1996-04-29 1996-04-29
US16487P 1996-04-29
GBGB9704161.0A GB9704161D0 (enrdf_load_stackoverflow) 1997-02-21 1997-02-21
GB9704161 1997-02-28
PCT/EP1997/001963 WO1997041221A1 (en) 1996-04-29 1997-04-18 Metastasis-associated antigen and antibodies thereto

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EP0906423A1 true EP0906423A1 (en) 1999-04-07

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EP (1) EP0906423A1 (enrdf_load_stackoverflow)
JP (1) JP2000515725A (enrdf_load_stackoverflow)
AU (1) AU2765697A (enrdf_load_stackoverflow)
GB (1) GB9704161D0 (enrdf_load_stackoverflow)

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JP5167813B2 (ja) * 2005-05-12 2013-03-21 味の素株式会社 タンパク質の製造法

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GB9704161D0 (enrdf_load_stackoverflow) 1997-04-16
AU2765697A (en) 1997-11-19
JP2000515725A (ja) 2000-11-28

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