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WO2000058460A2 - Casb619 involved in colon cancers - Google Patents

Casb619 involved in colon cancers Download PDF

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Publication number
WO2000058460A2
WO2000058460A2 PCT/EP2000/002478 EP0002478W WO0058460A2 WO 2000058460 A2 WO2000058460 A2 WO 2000058460A2 EP 0002478 W EP0002478 W EP 0002478W WO 0058460 A2 WO0058460 A2 WO 0058460A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
identity
cells
Prior art date
Application number
PCT/EP2000/002478
Other languages
English (en)
French (fr)
Other versions
WO2000058460A3 (en
Inventor
Claudine Elvire Marie Bruck
Jean-Pol Cassart
Thierry Coche
Carlota Vinals Y De Bassols
Original Assignee
Smithkline Beecham Biologicals S.A.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GBGB9907113.6A external-priority patent/GB9907113D0/en
Priority claimed from GBGB9922858.7A external-priority patent/GB9922858D0/en
Priority to IL14504800A priority Critical patent/IL145048A0/xx
Priority to EP00912627A priority patent/EP1165774A2/en
Priority to JP2000608741A priority patent/JP2002539826A/ja
Priority to HU0202930A priority patent/HUP0202930A2/hu
Priority to BR0009358-0A priority patent/BR0009358A/pt
Priority to CA002368014A priority patent/CA2368014A1/en
Application filed by Smithkline Beecham Biologicals S.A. filed Critical Smithkline Beecham Biologicals S.A.
Priority to MXPA01009688A priority patent/MXPA01009688A/es
Priority to AU34308/00A priority patent/AU3430800A/en
Priority to KR1020017012208A priority patent/KR20020008134A/ko
Publication of WO2000058460A2 publication Critical patent/WO2000058460A2/en
Publication of WO2000058460A3 publication Critical patent/WO2000058460A3/en
Priority to NO20014629A priority patent/NO20014629L/no
Priority to HK02104691.2A priority patent/HK1044961A1/zh

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to polynucleotides, herein referred to as CASB619 polynucleotides, polypeptides encoded thereby (referred to herein as CASB619 polypeptides), recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of cancer, in particular colon cancer and autoimmune diseases and other related conditions.
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with CASB619 polypeptide imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate CASB619 polypeptide activity or levels.
  • Polypeptides and polynucleotides of the present invention are believed to be important immunogens for specific prophylactic or therapeutic immunization against tumours, because they are specifically expressed or highly over-expressed in tumours compared to normal cells and can thus be targeted by antigen-specific immune mechanisms leading to the destruction of the tumour cell. They can also be used to diagnose the occurrence of tumour cells. Furthermore, their inappropriate expression in certain circumstances can cause an induction of autoimmune, inappropriate immune responses, which could be corrected through appropriate vaccination using the same polypeptides or polynucleotides. In this respect the most important biological activities to our purpose are the antigenic and immunogenic activities of the polypeptide of the present invention.
  • a polypeptide of the present invention may also exhibit at least one other biological activity of a CASB619 polypeptide, which could qualify it as a target for therapeutic or prophylactic intervention different from that linked to the immune response.
  • the present invention relates to CASB619 polypeptides.
  • Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • Such polypeptides include those comprising the amino acid of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • polypeptides include the polypeptide of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1.
  • the invention also provides an immunogenic fragment of a CASB619 polypeptide, that is a contiguous portion of the CASB619 polypeptide which has the same or similar immunogenic properties to the polypeptide comprising the amino acid seqeunce of SEQ ID NO:2. That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises the CASB619 polypeptide.
  • an immunogenic fragment may include, for example, the CASB619 polypeptide lacking an N- terminal leader sequence, a transmembrane domain or a C-terminal anchor domain.
  • the immunogenic fragment of CASB619 comprises substantially all of the extracellular domain of a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • Peptide fragments incorporating an epitope of CASB619 typically will comprise at least 7, preferably 9 or 10 contiguous amino acids from SEQ ID NO:2. Preferred epitopes are shown in SEQ ID NO:5 to SEQ ID NO:68.
  • Mimotopes which have the same characteristics as these epitopes, and immunogens comprising such mimotopes which generate an immune response which cross-react with an epitope in the context of the CASB619 molecule, also form part of the present invention.
  • the present invention therefore, includes isolated peptides encompassing these epitopes themselves, and any mimotope thereof.
  • the meaning of mimotope is defined as an entity which is sufficiently similar to the native CASB619 epitope so as to be capable of being recognised by antibodies which recognise the native molecule; (Gheysen, H.M., et al., 1986, Synthetic peptides as antigens.
  • Peptide mimotopes of the above-identified epitopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids.
  • the peptides of the present invention may be modified for the purposes of ease of conjugation to a protein carrier.
  • the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail.
  • the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide.
  • modified peptides could be a wholly or partly non-peptide mimotope wherein the constituent residues are not necessarily confined to the 20 naturally occurring amino acids.
  • these may be cyclised by techniques known in the art to constrain the peptide into a conformation that closely resembles its shape when the peptide sequence is in the context of the whole molecule.
  • a preferred method of cyclising a peptide comprises the addition of a pair of cysteine residues to allow the formation of a disulphide bridge.
  • mimotopes or immunogens of the present invention may be larger than the above-identified epitopes, and as such may comprise the sequences disclosed herein.
  • the mimotopes of the present invention may consist of addition of N and/or C terminal extensions of a number of other natural residues at one or both ends.
  • the peptide mimotopes may also be retro sequences of the natural sequences, in that the sequence orientation is reversed; or alternatively the sequences may be entirely or at least in part comprised of D-stereo isomer amino acids (inverso sequences).
  • the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are of the D-stereoisomer form.
  • retro or retro-inverso peptides have the advantage of being non-self, and as such may overcome problems of self-tolerance in the immune system.
  • peptide mimotopes may be identified using antibodies which are capable themselves of binding to the epitopes of the present invention using techniques such as phage display technology (EP 0 552 267 B 1).
  • This technique generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti -native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native peptide.
  • This approach may have significant advantages by allowing the possibility of identifying a peptide with enhanced immunogenic properties, or may overcome any potential self-antigen tolerance problems which may be associated with the use of the native peptide sequence. Additionally this technique allows the identification of a recognition pattern for each native-peptide in terms of its shared chemical properties amongst recognised mimotope sequences.
  • the covalent coupling of the peptide to the immunogenic carrier can be carried out in a manner well known in the art.
  • a carbodiimide, glutaraldehyde or (N-[ ⁇ -maleimidobutyryloxy] succinimide ester utilising common commercially available heterobifunctional linkers such as CDAP and SPDP (using manufacturers instructions).
  • the immunogen can easily be isolated and purified by means of a dialysis method, a gel filtration method, a fractionation method etc.
  • the types of carriers used in the immunogens of the present invention will be readily known to the man skilled in the art.
  • the function of the carrier is to provide cytokine help in order to help induce an immune response against the peptide.
  • carriers which may be used in the present invention include: Keyhole limpet Haemocyanin (KLH), serum albumins such as bovine serum albumin (BSA), inactivated bacterial toxins such as tetanus or diptheria toxins (TT and DT), or recombinant fragments thereof (for example, Domain 1 of Fragment C of TT, or the translocation domain of DT), or the purified protein derivative of tuberculin (PPD).
  • the mimotopes or epitopes may be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing T-cell help.
  • the ratio of mimotopes to carrier is in the order of 1 : 1 to 20: 1 , and preferably each carrier should carry between 3-15 peptides.
  • a preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 Bl).
  • Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted EP 0 594 610 Bl).
  • fragments of protein D for example Protein D l/3 rd (comprising the N-terminal 100-1 10 amino acids of protein D (GB 9717953.5)).
  • Another preferred method of presenting the peptides of the present invention is in the context of a recombinant fusion molecule.
  • EP 0 421 635 B describes the use of chimaeric hepadnavirus core antigen particles to present foreign peptide sequences in a virus-like particle.
  • immunogens of the present invention may comprise peptides presented in chimaeric particles consisting of hepatitis B core antigen.
  • the recombinant fusion proteins may comprise the mimotopes of the present invention and a carrier protein, such as NS1 of the influenza virus.
  • the nucleic acid which encodes said immunogen also forms an aspect of the present invention.
  • Peptides used in the present invention can be readily synthesised by solid phase procedures well known in the art. Suitable syntheses may be performed by utilising "T- boc" or "F-moc” procedures. Cyclic peptides can be synthesised by the solid phase procedure employing the well-known "F-moc” procedure and polyamide resin in the fully automated apparatus. Alternatively, those skilled in the art will know the necessary laboratory procedures to perform the process manually. Techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL at Oxford University Press (1989).
  • the peptides may be produced by recombinant methods, including expressing nucleic acid molecules encoding the mimotopes in a bacterial or mammalian cell line, followed by purification of the expressed mimotope.
  • Techniques for recombinant expression of peptides and proteins are known in the art, and are described in Maniatis, T., Fritsch, E.F. and Sambrook et al., Molecular cloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
  • polypeptides or immunogenic fragment of the invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
  • the invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
  • the proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non- fused protein.
  • the fusion partner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein.
  • the fusion partner will be both an immunological fusion partner and expression enhancing partner.
  • Fusion partners include protein D from Haemophilus influenza B and the non-structural protein from influenzae virus, NS 1 (hemagglutinin).
  • Another immunological fusion partner is the protein known as LYTA.
  • LYTA Preferably the C terminal portion of the molecule is used.
  • Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L- alanine amidase, amidase LYTA, (coded by the lytA gene ⁇ Gene, 43 (1986) page 265- 272 ⁇ an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
  • the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at its amino terminus has been described ⁇ Biotechnology: 10, (1992) page 795-798 ⁇ . It is possible to use the repeat portion of the Lyta molecule found in the C terminal end starting at residue 178, for example residues 188 - 305.
  • the present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination. Polypeptides of the present invention can be prepared in any suitable manner.
  • polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention relates to CASB619 polynucleotides.
  • polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90%> identity, yet more preferably at least 95%) identity, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
  • polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
  • polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO:2.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identiy are more highly preferred, and those with at least 99% identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 as well as the polynucleotide of SEQ ID NO: 1.
  • Said polynucleotide can be inserted in a suitable plasmid or recombinant microrganism vector and used for immunization (see for example Wolff et. al., Science 247:1465-1468 (1990); Corr et. al, J. Exp. Med. 184:1555-1560 (1996); Doe et. al., Proc. Natl. Acad. Sci. 93:8578-8583 (1996)).
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
  • the invention also provides a fragment of a CASB619 polynucleotide which when administered to a subject has the same immunogenic properties as the polynucleotide of SEQ ID NO: 1.
  • the invention also provides a polynucleotide encoding an immunological fragment of a CASB619 polypeptide as hereinbefore defined.
  • the nucleotide sequence of SEQ ID NO:l shows homology with Homo sapiens chromosome 1 clone RP4-641D22 map pi 3.1-13.3 (accession AL 157901).
  • the nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 3043) encoding a polypeptide of 1013 amino acids, the polypeptide of SEQ ID NO:2.
  • the nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:l or it may be a sequence other than the one contained in SEQ ID NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polypeptide of the SEQ ID NO:2 is notrelated to any other known protein.
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, ter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides, immunological fragments and polynucleotides of the present invention have at least one activity of either SEQ ID NO:l or SEQ ID NO:2, as appropriate.
  • the present invention also relates to partial or other incomplete polynucleotide and polypeptide sequences which were first identified prior to the determination of the corresponding full length sequences of SEQ ID NO:l and SEQ ID NO:2. Accordingly, in a further aspect, the present invention provides for an isolated polynucleotide which:
  • (a) comprises a nucleotide sequence which has at least 70% identity, preferably at least 80%) identity, more preferably at least 90% identity, yet more preferably at least 95%> identity, even more preferably at least 97-99% identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3;
  • (b) has a nucleotide sequence which has at least 70% identity, preferably at least 80%> identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity, to SEQ ID NO:l over the entire length of SEQ ID NO:3;
  • nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4; as well as the polynucleotide of SEQ ID NO:3.
  • the present invention further provides for a polypeptide which:
  • (a) comprises an amino acid sequence which has at least 70%> identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95%> identity, most preferably at least 97-99%> identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:4;
  • (b) has an amino acid sequence which is at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99%) identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:4;
  • (c) comprises the amino acid of SEQ ID NO:4;
  • (d) is the polypeptide of SEQ ID NO:4; as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:3.
  • nucleotide sequence of SEQ ID NO:3 and the peptide sequence encoded thereby are derived from EST (Expressed Sequence Tag) sequences. It is recognised by those skilled in the art that there will inevitably be some nucleotide sequence reading errors in EST sequences (see Adams, M.D. et al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded therefrom are therefore subject to the same inherent limitations in sequence accuracy. Furthermore, the peptide sequence encoded by SEQ ID NO: 3 comprises a region of identity or close homology and/or close structural similarity (for example a conservative amino acid difference) with the closest homologous or structurally similar protein.
  • Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human colon cancer, (for example Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring harbor Laboratory Press, Cold Spring harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non- translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polypeptide variants which comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SEQ ID NO: 1.
  • these nucleotide sequences are 70%> identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent.
  • the probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.
  • polypeptides or polynucleotides derived from sequences from homologous animal origin could be used as immunogens to obtain a cross-reactive immune response to the human gene.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Such hybridization techniques are well known to the skilled artisan.
  • Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C.
  • the present invention also includes polynucleotides obtainable by screening an appropriate library under stingent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is short at the 5' end of the cDNA.
  • RACE Rapid Amplification of cDNA ends
  • cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end.
  • Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers.
  • the PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence).
  • the products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to an expression system which comprises a polynucleotide of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • the proteins of the invention are coexpressed with thioredoxin in trans (TIT).
  • TIT thioredoxin in trans
  • Coexpression of thioredoxin in trans versus in cis is preferred to keep antigen free of thioredoxin without the need for protease.
  • Thioredoxin coexpression eases the solubilisation of the proteins of the invention.
  • Thioredoxin coexpression has also a significant impact on protein purification yield, on purified-protein solubility and quality.
  • bacterial cells such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as Streptococci, Staphylococci, E.
  • expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual (supra).
  • Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the expression system may also be a recombinant live microorganism, such as a virus or bacterium.
  • the gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses.
  • Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Dialoguelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
  • poxviruses e.g; vaccinia, fowlpox, canarypox
  • alphaviruses Semliki Forest Virus, Kunststoffuelian Equine Encephalitis Virus
  • adenoviruses adeno-associated virus
  • picornaviruses
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, ion metal affinity chromatography (IMAC) is employed for purification.
  • IMAC ion metal affinity chromatography
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and or purification.
  • Another important aspect of the invention relates to a method for inducing , re-inforcing or modulating an immunological response in a mammal which comprises inoculating the mammal with a fragment or the entire polypeptide or polynucleotide of the invention, adequate to produce antibody and/or T cell immune response for prophylaxis or for therapeutic treatment of cancer and autoimmune disease and related conditions.
  • Yet another aspect of the invention relates to a method of inducing, re-inforcing or modulating immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector or cell directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce immune responses for prophylaxis or treatment of said mammal from diseases.
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces, re-inforces or modulates an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the invention or an immunological fragment thereof as herein before defined.
  • the vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • a further aspect of the invention relates to the in vitro induction of immune responses to a fragment or the entire polypeptide or polynucleotide of the present invention or a molecule comprising the polypeptide or polynucleotide of the present invention, using cells from the immune system of a mammal, and reinfusing these activated immune cells of the mammal for the treatment of disease.
  • Activation of the cells from the immune system is achieved by in vitro incubation with the entire polypeptide or polynucleotide of the present invention or a molecule comprising the polypeptide or polynucleotide of the present invention in the presence or absence of various immunomodulator molecules.
  • a further aspect of the invention relates to the immunization of a mammal by administration of antigen presenting cells modified by in vitro loading with part or the entire polypeptide of the present invention or a molecule comprising the polypeptide of the present invention and administered in vivo in an immunogenic way.
  • antigen presenting cells can be transfected in vitro with a vector containing a fragment or the entire polynucleotide of the present invention or a molecule comprising the polynucleotide of the present invention, such as to express the corresponding polypeptide, and administered in vivo in an immunogenic way.
  • the vaccine formulation of the invention may also include adjuvant systems for enhancing the immunogenicity of the formulation.
  • the adjuvant system raises preferentially a TH1 type of response.
  • An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed THl-type responses (cell-mediated response), and TH2-type immune responses (humoral response).
  • Extreme THl-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses.
  • mice THl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies.
  • TH2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
  • cytokines the driving force behind the development of these two types of immune responses.
  • High levels of THl-type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
  • TH1 and TH2-type immune responses are not absolute. In reality an individual will support an immune response which is described as being predominantly TH1 or predominantly TH2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 +ve T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989) TH1 and TH2 cells: different patterns oflymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, pi 45-173). Traditionally, THl-type responses are associated with the production of the INF- ⁇ and IL-2 cytokines by T-lymphocytes.
  • cytokines often directly associated with the induction of THl-type immune responses are not produced by T-cells, such as IL-12.
  • TH2- type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.
  • the best indicators of the TH1 :TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of TH1 or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses.
  • a THl-type adjuvant is one which preferentially stimulates isolated T-cell populations to produce high levels of THl-type cytokines when re-stimulated with antigen in vitro, and promotes development of both CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulin responses associated with THl-type isotype.
  • Adjuvants which are capable of preferential stimulation of the TH1 cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.
  • 3 De-O-acylated monophosphoryl lipid A is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 Bl (SmithKline Beecham Biologicals SA).
  • the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (European Patent number 0 689 454).
  • 3D-MPL will be present in the range of lO ⁇ g - lOO ⁇ g preferably 25-50 ⁇ g per dose wherein the antigen will typically be present in a range 2-50 ⁇ g per dose.
  • Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina. Optionally this may be admixed with 3 De- O-acylated monophosphoryl lipid A (3D-MPL), optionally together with an carrier.
  • Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful TH1 stimulating adjuvants when formulated together with an antigen.
  • Further adjuvants which are preferential stimulators of TH1 cell response include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
  • TH1 stimulating adjuvants such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of TH1 cell response.
  • QS21 can be formulated together with 3D- MPL.
  • the ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1; preferably 1 :5 to 5 : 1 and often substantially 1 : 1.
  • the preferred range for optimal synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
  • a carrier is also present in the vaccine composition according to the invention.
  • the carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.
  • a preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tween 80.
  • a metabolisible oil such as squalene, alpha tocopherol and Tween 80.
  • the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion.
  • the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
  • QS21 and 3D-MPL will be present in a vaccine in the range of l ⁇ g - 200 ⁇ g, such as 10-100 ⁇ g, preferably lO ⁇ g - 50 ⁇ g per dose.
  • the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80.
  • the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion.
  • Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
  • Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier.
  • a non-toxic oil e.g. squalane or squalene
  • an emulsifier e.g. Tween 80
  • the aqueous carrier may be, for example, phosphate buffered saline.
  • the present invention also provides a polyvalent vaccine composition
  • a polyvalent vaccine composition comprising a vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating cancers, autoimmune diseases and related conditions.
  • a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.
  • This invention also relates to the use of polynucleotides, in the form of primers derived from the polynucleotides of the present invention, and of polypeptides, in the form of antibodies or reagents specific for the polypeptide of the present invention, as diagnostic reagents.
  • the identification of genetic or biochemical markers in blood or tissues that will enable the detection of very early changes along the carcinogenesis pathway will help in determining the best treatment for the patient.
  • Surrogate tumour markers such as polynucleotide expression, can be used to diagnose different forms and states of cancer.
  • the identification of expression levels of the polynucleotides of the invention will be useful in both the staging of the cancerous disorder and grading the nature of the cancerous tissue.
  • the staging process monitors the advancement of the cancer and is determined on the presence or absence of malignant tissue in the areas biopsied.
  • the polynucleotides of the invention can help to perfect the staging process by identifying markers for the aggresivity of a cancer, for example the presence in different areas of the body.
  • the grading of the cancer describes how closely a tumour resembles normal tissue of its same type and is assessed by its cell morphology and other markers of differentiation.
  • the polynucleotides of the invention can be useful in determining the tumour grade as they can help in the determination of the differentiation status of the cells of a tumour.
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to cancers, autoimmune disease and related conditions through diagnosis by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA.
  • This method of diagnosis is known as differential expression.
  • the expression of a particular gene is compared between a diseased tissue and a normal tissue.
  • a difference between the polynucleotide-related gene, mRNA, or protein in the two tissues is compared, for example in molecular weight, amino acid or nucleotide sequence, or relative abundance, indicates a change in the gene, or a gene which regulates it, in the tissue of the human that was suspected of being diseased.
  • RNA level Decreased or increased expression can be measured at the RNA level.
  • PolyA RNA is first isolated from the two tissues and the detection of mRNA encoded by a gene corresponding to a differentially expressed polynucleotide of the invention can be detected by, for example, in situ hybridization in tissue sections, reverse trascriptase- PCR, using Northern blots containing poly A+ mRNA, or any other direct or inderect RNA detection method.
  • An increased or decreased expression of a given RNA in a diseased tissue compared to a normal tissue suggests that the transcript and/or the expressed protein has a role in the disease.
  • detection of a higher or lower level of mRNA corresponding to SEQ ID NO 1 or 3 relative to normal level is indicative of the presence of cancer in the patient.
  • mRNA expression levels in a sample can be determined by generation of a library of expressed sequence tags (ESTs) from the sample.
  • ESTs expressed sequence tags
  • the relative representation of ESTs in the library can be used to assess the relative representation of the gene transcript in the starting sample.
  • the EST analysis of the test can then be compared to the EST analysis of a reference sample to determine the relative expression levels of the polynucleotide of interest.
  • Other mRNA analyses can be carried out using serial analysis of gene expression (SAGE) methodology (Velculescu et. Al. Science (1995) 270:484) , differential display methodology (For example, US 5,776,683) or hybridization analysis which relies on the specificity of nucleotide interactions.
  • the comparison could be made at the protein level.
  • the protein sizes in the two tissues may be compared using antibodies to detect polypeptides in Western blots of protein extracts from the two tissues. Expression levels and subcellular localization may also be detected immunologically using antibodies to the corresponding protein. Further assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. A raised or decreased level of polypeptide expression in the diseased tissue compared with the same protein expression level in the normal tissue indicates that the expressed protein may be involved in the disease.
  • the diagnosis can be determined by detection of gene product expression levels encoded by at least one sequence set forth in SEQ ID NOS: 1 or 3.
  • a comparison of the mRNA or protein levels in a diseased versus normal tissue may also be used to follow the progression or remission of a disease.
  • polynucleotide sequences in a sample can be assayed using polynucleotide arrays. These can be used to examine differential expression of genes and to determine gene function. For example, arrays of the polynucleotide sequences SEQ ID NO: 1 or 3 can be used to determine if any of the polynucleotides are differentially expressed between a normal and cancer cell.
  • an array of oligonucleotides probes comprising the SEQ ID NO: 1 or 3 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Diagnosis includes determination of a subject's susceptibility to a disease, determination as to whether a subject presently has the disease, and also the prognosis of a subject affected by the disease.
  • the present invention further relates to a diagnostic kit for performing a diagnostic assay which comprises:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1 or 3, or a fragment thereof ;
  • the nucleotide sequences of the present invention are also valuable for chromosomal localisation.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined.
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for polypeptides of the present invention.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • the invention provides an antibody immunospecific for a polypeptide according to the invention or an immunological fragment thereof as hereinbefore defined.
  • the antibody is a monoclonal antibody
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole etal, Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).
  • Patent No. 4,946,778 can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • the antibody of the invention may also be employed to prevent or treat cancer, particularly ovarian and colon cancer, autoimmune disease and related conditions.
  • Another aspect of the invention relates to a method for inducing or modulating an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and or T cell immune response to protect or ameliorate the symptoms or progression of the disease.
  • Yet another aspect of the invention relates to a method of inducing or modulating immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • the present invention therefore provides a method of treating abnormal conditions such as, for instance, cancer and autoimmune diseases, in particular, ovarian and colon cancer, related to either a presence of, an excess of, or an under- expression of, CASB619 polypeptide activity.
  • abnormal conditions such as, for instance, cancer and autoimmune diseases, in particular, ovarian and colon cancer, related to either a presence of, an excess of, or an under- expression of, CASB619 polypeptide activity.
  • the present invention further provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the CASB619 polypeptide.
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such diseases as hereinbefore mentioned.
  • Compounds may be identified from a variety of sources, for example, cells, cell- free preparations, chemical libraries, and natural product mixtures.
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991)). Screening methods will be known to those skilled in the art.
  • the invention provides a method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of the invention which comprises a method selected from the group consisting of:
  • the polypeptide of the invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. Well known screening methods may also be used to identify agonists and antagonists of the polypeptide of the invention which compete with the binding of the polypeptide of the invention to its receptors, if any.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:
  • polypeptide of the present invention (c) a cell membrane expressing a polypeptide of the present invention; or (d) antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2.
  • polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:
  • Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by FuUerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
  • each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise l-1000 ⁇ g of protein, preferably 2-100 ⁇ g, most preferably 4-40 ⁇ g. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA including single and double stranded regions.
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol.
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al. , NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • the preferred algorithm used is FASTA.
  • the preferred parameters for polypeptide or polynuleotide sequence comparison using this algorithm include the following: Gap Penalty: 12 Gap extension penalty: 4 Word size: 2, max 6
  • Preferred parameters for polypeptide sequence comparison with other methods include the following:
  • Gap Length Penalty 3 A program useful with thesejiarameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for polynucleotide comparisons.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l, that is be 100%) identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
  • Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: l by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO:l, or: n n ⁇ x n - (x n • y), wherein n n is the number of nucleotide alterations, x n is the total number of nucleotides in SEQ ID NO:l, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n a ⁇ x a " (*a • y)> wherein n a is the number of amino acid alterations, x a is the total number of amino acids in SEQ ID NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quntified by determining the degree of identity and/or similarity between the sequences being compared as hereinbefore described. Falling within this generic term are the terms “ortholog”, meaning a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species and "paralog” meaning a functionally similar sequence when considered within the same species.
  • Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) is used to compare mRNA transcript abundance of the candidate antigen in matched tumour and normal colon tissues from multiple patients. In addition, mRNA levels of the candidate gene in a panel of normal tissues are evaluated by this approach.
  • Total RNA from normal and tumour colon is extracted from snap frozen biopsies using TriPure reagent (Boehringer).
  • Total RNA from normal tissues is purchased from InVitrogen or is extracted from snap frozen biopsies using TriPure reagent.
  • Poly-A+ mRNA is purified from total RNA after DNAase treatment using oligo-dT magnetic beads (Dynal). Quantification of the mRNA is performed by spectrofluorimetry (VersaFluor, BioRad) using Sybrll dye (Molecular Probes). Primers for real-time PCR amplification are designed with the Perkin-Elmer Primer Express software using default options for TaqMan amplification conditions.
  • Real-time reactions are assembled according to standard PCR protocols using 2 ng of purified mRNA for each reaction.
  • Sybrl dye (Molecular Probes) is added at a final dilution of 1/75000 for real-time detection.
  • Amplification (40 cycles) and real-time detection is performed in a Perkin-Elmer Biosystems PE7700 system using conventional instrument settings.
  • Ct values are calculated using the PE7700 Sequence Detector software. Two Ct values are obtained for each patient sample: the tumour Ct (CtT) and the matched normal colon Ct (CtN).
  • Ct values obtained by real-time PCR are log-linearly related to the copy number of the target template.
  • CtN-CtT is an estimate of the relative transcript levels in the two tissues (i.e. fold mRNA over-expression in tumor).
  • Real-time PCR reactions are performed on biopsies from 24 patients. The level of mRNA over-expression is calculated as described for each patient, average level of mRNA over-expression for the candidate antigen and the proportion of patients over-expressing the candidate antigen is then calculated from this data set. The individual values are standardised with respect to actin in the same sample (ratio), as seen in Figure 1. A value of 1 thus corresponds to the same level of actin expression. The results are shown in a logarithmic scale.
  • CASB619 is overexpressed in 67% of colon cancer samples with respect to the adjacent normal colon, at an average rate of nearly 200 fold.
  • the expression in normal tissues is restricted to other digestive tract tissues, as well as mainly trachea and testis.
  • D ⁇ A micro-arrays are used to examine mR ⁇ A expression profiles of large collections of genes in multiple samples. This information is used to complement the data obtained by real-time PCR and provides an independent measure of gene expression levels in tumors and normal tissues.
  • Examples of current technologies for production of D ⁇ A micro-arrays include 1 ) The Affymetrix "GeneChip" arrays in which oligonucleotides are synthetized on the surface of the chip by solid phase chemical synthesis using a photolithographic process 2) D ⁇ A spotting technology in which small volumes of a D ⁇ A solution are robotically deposited and then immobilized onto the surface of a solid phase (e.g. glass). In both instances, the chips are hybridized with cD ⁇ A or cR ⁇ A which has been extracted from the tissue of interest (e.g. normal tissue, tumour etc ..) and labeled with radioactivity or with a fluorescent reporter molecule.
  • tissue of interest e.g. normal tissue, tumour etc ..
  • the labeled material is hybridized to the chip and the amount of probe bound to each sequence on the chip is determined using a specialized scanner.
  • the experiment can be set-up with a single fluorescent reporter (or radioactivity) or, alternatively, can be performed using two fluorescent reporters. In this latter case, each of the two samples is labeled with one of the reporter molecules.
  • the two labeled samples are then hybridized competitively to the sequences on the D ⁇ A chip.
  • the ratio of the two fluorescent signals is determined for each sequence on the chip. This ratio is used to calculate the relative abundance of the transcript in the two samples.
  • Detailed protocols are available from a number of sources including "D ⁇ A Microarrays: A practical approach. Schena M.
  • ESTs are small fragments of cDNA made from a collection of mRNA extracted from a particular tissue or cell line.
  • Such database currently provide a massive amount of ESTs (106) from several hundreds of cDNA tissue libraries, including tumoral tissues from various types and states of disease.
  • Blast informatics tools
  • NCBM737305 895 ' NCI_CGAP_Br2 (pooled breast tumor tissues)
  • NCBI:2381271 1461 NC ⁇ _CGAP_Ut1
  • Northern blots are produced according to standard protocols using 1 ⁇ g of poly A+ mRNA. Radioactive probes are prepared using the Ready -to-Go system (Pharmacia).
  • Colon tumour cDNA libraries are constructed using the Lambda Zap II system (Stratagene) from 5 ⁇ g of polyA+ mRNA. The supplied protocol is followed except that Superscriptll (Life Technologies) is used for the reverse transcription step. Oligo dT- primed and random-primed libraries are constructed. About 1.5 x 106 independent phages are plated for each screening of the library. Phage plaques are transferred onto nylon filters and hybridised using a cDNA probe labelled with AlkPhos Direct. Positive phages are detected by chemiluminescence. Positive phage are excised from the agar plat, eluted in 500 ⁇ l SM buffer and confirmed by gene-specific PCR.
  • Eluted phages are converted to single strand Ml 3 bacteriophage by in vivo excision.
  • the bacteriophage is then converted to double strand plasmid DNA by infection of E. coli.
  • Infected bacteria are plated and submitted to a second round of screening with the cDNA probe. Plasmid DNA is purified from positive bacterial clones and sequenced on both strands. When the full length gene cannot be obtained directly from the cDNA library, missing sequence is isolated using RACE technology (Marathon Kit, ClonTech.).
  • This approach relies on reverse transcribing mRNA into double strand cDNA, ligating linkers onto the ends of the cDNA and amplifying the desired extremity of the cDNA using a gene- specific primer and one of the linker oligonucleotides.
  • Marathon PCR products are cloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.
  • the obtained sequence (SEQ ID NO:l) has a putative open reading frame of 1013 amino acids (SEQ ID NO:2).
  • the deduced protein sequence was submitted to prediction algorithms for cellular localisation (PSORT: http://psort.nibb.ac.jp/ and TopPred: http://www.biokemi.su.se/ ⁇ server/toppred2/toppred_source.html).
  • CASB619 seems to have a peptide signal, and one to three transmembrane domains (low confidence prediction). No other motif or domain was found.
  • Expression in microbial hosts, or alternatively in vitro transcription/translation, is used to produce the antigen of the invention for vaccine purposes and to produce protein fragments or whole protein for rapid purification and generation of antibodies needed for characterization of the naturally expressed protein by immunohistochemistry or for follow-up of purification.
  • Recombinant proteins may be expressed in two microbial hosts, E. coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris). Pichia. This allows the selection of the expression system with the best features for this particular antigen production. In general, the recombinant antigen will be expressed in E. coli and the reagent protein expressed in yeast.
  • the expression strategy first involves the design of the primary structure of the recombinant antigen.
  • an expression fusion partner (EFP) is placed at the N terminal extremity to improve levels of expression that could also include a region useful for modulating the immunogenic properties of the antigen, an immune fusion partner (IFP).
  • an affinity fusion partner (AFP ) useful for facilitating further purification is included at the C-terminal end.
  • the recombinant product is characterized by the evaluation of the level of expression and the prediction of further solubility of the protein by analysis of the behavior in the crude extract.
  • a comparative evaluation of the different versions of the expressed antigen will allow the selection of the most promising candidate that is to be used for further purification and immunological evaluation.
  • the purification scheme follows a classical approach based on the presence of an His affinity tail in the recombinant protein.
  • the disrupted cells are filtered and the acellular extracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni++NTA from Qiagen) that will specifically retain the recombinant protein.
  • IMAC Ion Metal Affinity Chromatography
  • the retained proteins are eluted by 0-500 mM Imidazole gradient (possibly in presence of a detergent) in a phosphate buffer.
  • Antibody production and immunohistochemistry Small amounts of relatively purified protein can be used to generate immunological tools in order to a) detect the expression by immunohistochemistry in normal or cancer tissue sections; b) detect the expression, and to follow the protein during the purification process (ELISA/ Western Blot); or c) characterise/ quantify the purified protein (ELISA).
  • ELISA 96 well microplates (maxisorb Nunc) are coated with 5 ⁇ g of protein overnight at 4°C. After lhour saturation at 37°C with PBS NCS 1%, serial dilution of the rabbit sera is added for 1H 30 at 37°C (starting at 1/10). After 3 washings in PBS Tween, anti rabbit biotinylated anti serum (Amersham ) is added (1/5000). Plates are washed and peroxydase coupled streptavidin (1/5000) is added for 30 min at 37°C. After washing, 50 ⁇ l TMB (BioRad) is added for 7 min and the reaction then stopped with H2SO4 0.2M. The OD can be measured at 450 nm and midpoint dilutions calculated by SoftmaxPro.
  • Spleen cells are fused with the SP2/0 myeloma according to a standard protocol using PEG 40%o and DMSO 5%. Cells are then seeded in 96 well plates 2.5 x 104 - 105 cells/well and resistant clones will be selected in HAT medium. The supernatant of these hybridomas will be tested for their content in specific antibodies and when positive, will be submitted to 2 cycles of limited dilution . After 2 rounds of screening, 3 hybridomas will be chosen for ascitis production.
  • immuno staining is performed on normal or cancer tissue sections, in order to determine :
  • tissue sample preparation After dissection, the tissue sample is mounted on a cork disk in OCT compound and rapidly frozen in isopentane previously super cooled in liquid nitrogen (-160°C). The block will then be conserved at -70°C until use. 7-10 ⁇ m sections will be realised in a cryostat chamber (-20, -30°C).
  • Tissue sections are dried for 5 min at room Temperature (RT), fixed in acetone for lOmin at RT, dried again, and saturated with PBS 0.5% BSA 5% serum. After 30 min at RT either a direct or indirect staining is performed using antigen specific antibodies. A direct staining leads to a better specificity but a less intense staining whilst an indirect staining leads to a more intense but less specific staining.
  • T cell lymphocyte lines and dendritic cells are derived from PBMCs (peripheral blood mononuclear cells) of healthy donors (preferred HLA-A2 subtype).
  • PBMCs peripheral blood mononuclear cells
  • HLA-A2.1/Kb transgenic mice is also used for screening of HLA-A2.1 peptides.
  • Newly discovered antigen-specific CD8+ T cell lines are raised and maintained by weekly in vitro stimulation.
  • the lytic activity and the D-IFN production of the CD 8 lines in response to the antigen or antigen derived-peptides is tested using standard procedures.
  • HLA-A2 binding peptide sequences are predicted either by the Parker's algorithm (Parker, K. C, M. A. Bednarek, and J. E. Coligan. 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side- chains. J. Immunol. 152: 163 and http://bimas.dcrt.nih.gov/molbio/hla_bind/) or the
  • transgenic mice are immunized with adjuvanted HLA-A2 peptides, those unable to induce a CD8 response (as defined by an efficient lysis of peptide-pulsed autologous spleen cells) will be further analyzed in the human system.
  • Human dendritic cells (cultured according to Romani et al.) will be pulsed with peptides and used to stimulate CD8-sorted T cells (by Facs). After several weekly stimulations, the CD8 lines will be first tested on peptide-pulsed autologous BLCL (EBV-B transformed cell lines). To verify the proper in vivo processing of the peptide, the CD8 lines will be tested on cDNA-transfected tumour cells (HLA-A2 transfected LnCaP, Skov3 or CAMA tumour cells).
  • cDNA-transfected tumour cells HLA-A2 transfected LnCaP, Skov3 or CAMA tumour cells.
  • CD8+ T cell lines will be primed and stimulated with either gene-gun transfected dendritic cells, retrovirally transduced B7.1 -transfected fibroblasts, recombinant pox virus (Kim et al.) or adenovirus (Butterfield et al.) infected dendritic cells.
  • Virus infected cells are very efficient to present antigenic peptides since the antigen is expressed at high level but can only be used once to avoid the over-growth of viral T cells lines.
  • CD8+ lines are tested on cDNA-transfected tumour cells as indicated above. Peptide specificity and identity is determined to confirm the immunological validation.

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AU34308/00A AU3430800A (en) 1999-03-26 2000-03-20 Novel compounds
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MXPA01009688A MXPA01009688A (es) 1999-03-26 2000-03-20 Compuestos novedosos.
JP2000608741A JP2002539826A (ja) 1999-03-26 2000-03-20 新規化合物
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BR0009358-0A BR0009358A (pt) 1999-03-26 2000-03-20 Compostos
CA002368014A CA2368014A1 (en) 1999-03-26 2000-03-20 Casb619 involved in colon cancers
IL14504800A IL145048A0 (en) 1999-03-26 2000-03-20 Casb619 involved in colon cancers
EP00912627A EP1165774A2 (en) 1999-03-26 2000-03-20 Casb619 involved in colon cancers
NO20014629A NO20014629L (no) 1999-03-26 2001-09-24 Nye forbindelser
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WO2001031003A1 (fr) * 1999-10-29 2001-05-03 Pierre Fabre Medicament Clonage, expression et caracterisation d'un gene exprime dans des cellules tumorales et implique dans la regulation de la reponse immune
WO2003072779A1 (fr) * 2002-02-27 2003-09-04 Japan Science And Technology Agency Methode d'utilisation de genes specifiques a l'hypophyse
WO2004085666A2 (en) * 2003-03-20 2004-10-07 Incyte Corporation Secreted proteins
US6951738B2 (en) 1999-07-16 2005-10-04 Human Genome Sciences, Inc. Human tumor necrosis factor receptors TR13 and TR14
US7291700B2 (en) 2000-06-05 2007-11-06 Genentech, Inc. PRO4985 polypeptide

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WO2004058153A2 (en) * 2002-12-20 2004-07-15 Millennium Pharmaceuticals, Inc. Methods and compositions for treating cancer using 15986, 2188, 20743, 9148, 9151, 9791, 44252, 14184, 42461, 8204, 7970, 25552, 21657, 26492, 2411, 15088, 1905, 28899, 63380, 33935, 10480, 12686, 25501, 17694, 15701, 53062, 49908, 21612, 38949, 6216, 46863, 9235, 2201, 6985, 9883, 12238, 18057, 21617, 39228, 49928, 54476.

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951738B2 (en) 1999-07-16 2005-10-04 Human Genome Sciences, Inc. Human tumor necrosis factor receptors TR13 and TR14
WO2001031003A1 (fr) * 1999-10-29 2001-05-03 Pierre Fabre Medicament Clonage, expression et caracterisation d'un gene exprime dans des cellules tumorales et implique dans la regulation de la reponse immune
US7291700B2 (en) 2000-06-05 2007-11-06 Genentech, Inc. PRO4985 polypeptide
WO2003072779A1 (fr) * 2002-02-27 2003-09-04 Japan Science And Technology Agency Methode d'utilisation de genes specifiques a l'hypophyse
WO2004085666A2 (en) * 2003-03-20 2004-10-07 Incyte Corporation Secreted proteins
WO2004085666A3 (en) * 2003-03-20 2005-07-07 Incyte Corp Secreted proteins

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