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WO2003016344A2 - Nouvelle utilisation - Google Patents

Nouvelle utilisation Download PDF

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Publication number
WO2003016344A2
WO2003016344A2 PCT/EP2002/009006 EP0209006W WO03016344A2 WO 2003016344 A2 WO2003016344 A2 WO 2003016344A2 EP 0209006 W EP0209006 W EP 0209006W WO 03016344 A2 WO03016344 A2 WO 03016344A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
lung cancer
cells
sequence
Prior art date
Application number
PCT/EP2002/009006
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English (en)
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WO2003016344A3 (fr
Inventor
Swann Romain Jean-Thomas Gaulis
Carlota Vinals Y De Bassols
Original Assignee
Glaxosmithkline Biologicals S.A.
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Publication date
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Priority to AU2002331231A priority Critical patent/AU2002331231A1/en
Publication of WO2003016344A2 publication Critical patent/WO2003016344A2/fr
Publication of WO2003016344A3 publication Critical patent/WO2003016344A3/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3023—Lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods for using polypeptides and polynucleotides (herein referred to as “CASB933 " polypeptide(s) and “ CASB933 " polynucleotide(s)”), including the treatment of cancer and autoimmune diseases, more particularly preneoplasic lesions of lung cancer and lung cancer.
  • CASB933 polypeptides and polynucleotides find utility in the treatment of small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or IIB of Mountain's staging) NSCLC, and other related conditions.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung cancer
  • the invention relates to pharmaceutical compositions containing CASB933 polypeptides and polynucleotides, to methods of manufacture of such compositions and to their use in medicine.
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with CASB933 polypeptide imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting preneoplasic lesions of lung cancer and lung cancer, more particularly for detecting small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) as listed herein above, and other related conditions associated with inappropriate CASB933 polypeptide activity or levels.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung cancer
  • CASB933 encodes a polypeptide which is known as Homo Sapiens E74-like factor 3 (ets domain transcription factor, epithelial-specific) (GenPept accession number AAH03569), or epithelium-restricted Ets protein ESX (GenPept accession number AFl 10184), or epthelial-specific ets protein (GenPept accession number AAC51884), or Ets transcription factor (GenPept accession number AAB96586), or Ets- related transcription factor (GenPept accession number AAB67238), or ESE-lb GenPept accession number AAB65823), or ESX (GenPept accession number AAB58075).
  • E74-like factor 3 ets domain transcription factor, epithelial-specific
  • ESX epithelium-restricted Ets protein ESX
  • GenPept accession number AAC51884 epithelium-restricted Ets protein
  • Ets protein
  • ELF3 Human E74-like factor 3 was isolated and charaterised as a novel member of the ETS transcription factor family in 1997.
  • ELF3 was shown to be exclusively expressed in epithelial cells, as well as induced during terminal differentiation of the epidermis and in a primary human keratinocyte differentiation system, suggesting a critical role for as a regulator of epithelial cell differentiation (Oettgen P, et al. Isolation and caraterization of a novel epithehuni-specific transcription factor, ESE-1, a member of the Ets family. Molec Cell Biol 17, 4419-4433, 1997).
  • ELF3 locus was localised to locus lq32, a genomic region that is frequently amplified in epithelial tumours (Tymms MJ, et al., 1997, Oncogene 15, 2449-2462; Chang CH, et al., 1997) and for example tumours of breast (Oncogene 14, 1617- 16221) and also prostate.
  • the invention relates to the use of CASB933 polypeptides and polynucleotides as described in greater detail below.
  • the invention relates especially to the use of CASB933 polynucleotides having the nucleotide set out in SEQ ID NO:l or SEQ ID NO:5 or SEQ ID NO:7.
  • the invention further relates to uses of polynucleotides which have at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity over the entire length of SEQ ID NO:l or SEQ ID NO:5 or SEQ ID NO:7.
  • a still most preferred embodiment is the use of CASB933 polynucleotides sequence set out in SEQ ID NO:l or SEQ ID NO:5 or SEQ ID NO:7.
  • the invention in a second embodiment, relates to the use of CASB933 polypeptides having the nucleotide set out in SEQ ID NO:2 or SEQ ID NO:6.
  • the invention further relates to uses of polypeptides which have at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity over the entire length of SEQ ID NO:2 or SEQ ID NO:6.
  • a still more preferred embodiment is the use of CASB933 polypeptides having the sequence set out in SEQ ID NO:2 or SEQ ID NO:6.
  • CASB933 polypeptides and polynucleotides of the present invention are believed to be important immunogens for specific prophylactic or therapeutic immunisation against tumours, more particularly preneoplasic lesions of lung tumours and lung tumours, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesotheliomas, even more particularly NSCLC preneoplasic lesion tumours and early stage (classification stages 0, or IA, or IB, or IIA, or IIB of Mountain's staging) NSCLC, because they are specifically expressed or highly over-expressed in tumours for instance such as above-mentionned tumours, compared to normal cells and can thus be targeted by antigen- specific immune mechanisms leading to the destruction of the tumour cell
  • a polypeptide for use according to the present invention may also exhibit at least one other biological activity of a CASB933 polypeptide, which could qualify it as a target for therapeutic or prophylactic intervention different from that linked to the immune response.
  • CASB933 polypeptides as well as biologically, diagnostically, prophylatically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • the invention further provides uses for:
  • polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:l or 5 or 7 «PN_SEQID» over the entire length of SEQ ID NO:l or 5 or 7 respectively «PN_SEQID»; or
  • polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97- 99% or exact identity, to the amino acid sequence of SEQ ID NO:2 or 6 over the entire length of SEQ ID NO:2 or 6 respectively «PP_SEQID».
  • the invention also provides an immunogenic fragment of a CASB933 polypeptide, that is a contiguous portion of the CASB933 polypeptide which has the same or similar immunogenic properties to the polypeptide comprising the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:6. That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises the CASB933 polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of SEQ H NO:2 «PP_SEQID» or 6 or of a variant thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, are also preferred.
  • fragments characterized by structural or functional attributes such as fragments that comprise beta-barrels, alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet-foirning regions, turn and turn-forming regions, coil and coil-forming regions, hydrophiUc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • an immunogenic fragment may include, for example, the CASB933 polypeptide lacking an N-terminal leader sequence, a transmembrane domain or a C-terminal anchor domain.
  • the immunogenic fragment of CASB933 according to the invention 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 and SEQ ID NO:6 over the entire length of SEQ ID NO:2 and SEQ ID NO:6.
  • an immunogenic fragment for use according to the invention comprises at least one epitope.
  • Peptide fragments incorporating an epitope of CASB933 typically will comprise at least 7, preferably 9 or 10 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:6.
  • Preferred epitopes are shown in SEQ TD NO:8 to SEQ ID NO:75. Peptides that incorporate these epitopes form a preferred aspect of the present invention.
  • the fragments have a level of immunogenic activity of at least about 50%, preferably at least about 70% and more preferably at least about 90% of the level of immunogenic activity of a polypeptide sequence set forth in SEQ ID NO:2 and SEQ ID NO:6 or a polypeptide sequence encoded by a polynucleotide sequence set forth in SEQ ID NO:l and SEQ ID NO:5 and SEQ ID NO:7.
  • the invention also provides a fragment of a
  • CASB933 polynucleotide which when administered to a subject has the same immunogenic properties as the polynucleotide of SEQ ID NO:l and SEQ ID NO:5 and SEQ ID NO:7.
  • polypeptide fragments according to the invention preferably comprise at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide composition set forth herein, such as those set forth in SEQ ID NO:2 and SEQ ID NO:6, or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID NO:l and SEQ ID NO:5 and SEQ ID NO:7.
  • 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 CASB933 molecule, also form part of the present invention.
  • the present invention therefore, provides uses for isolated peptides encompassing these epitopes themselves, and any mimotope thereof.
  • mimotope is defined as an entity which is sufficiently similar to the native CASB933 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. Wiley, Chichester, Ciba foundation symposium 119, pl30-149; Gheysen, H.M., 1986, Molecular Immunology, 23,7, 709- 715); or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies cross-react with the native molecule.
  • 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. Accordingly, 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 maybe 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. Such 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 Bl).
  • 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.
  • a non-exhaustive list of carriers which may be used in the present invention include: Keyhole limpet
  • Haemocyanin KLH
  • serum albumins such as bovine serum albumin (BSA)
  • BSA bovine serum albumin
  • TT and DT inactivated bacterial toxins
  • recombinant fragments thereof for example, Domain 1 of Fragment C of TT, or the translocation domain of DT
  • PPD purified protein derivative of tuberculin
  • 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 100-110 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 0421 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.
  • polypeptides are recombinantly produced.
  • polypeptides according to the invention are purified, and are substantially free of any other proteins or contaminating host-originating material.
  • 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).
  • the polypeptides or immunogenic fragment for use according to the present 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.
  • the invention relates to genetically engineered soluble fusion proteins comprising a polypeptide as defined in the present invention, or a fragment thereof, and a carrier.
  • the carrier represents various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • an 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, NS1 (hemagglutinin).
  • Another immunological fusion partner is the protein known as LYTA.
  • LYTA the protein known as the protein known as LYTA.
  • 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 xenogeneic forms (also termed ortholog forms) of the aforementioned polypeptides, said xenogeneic forms referring to an antigen having substantial sequence identity to the human antigen (also termed autologous antigen) which serves as a reference antigen but which is derived from a different non-human species.
  • the substantial identity refers to concordance of an amino acid sequence with another amino acid sequence or of a polynucleotide sequence with another polynucleotide sequence when such sequence are arranged in a best fit alignment in any of a number of sequence alignment proteins known in the art.
  • the xenogeneic CASB933 polypeptide will be a CASB933 polypeptide which is xenogeneic with respect to human CASB933, in other words which is isolated from a species other than human.
  • the polypeptide is isolated from mouse, rat, pig, or rhesus monkey, most preferably from mouse or rat.
  • the present invention also provides a method of inducing an immune response against human CASB933 having an amino acid sequence as set forth in any of the sequences SEQ ID NO:3 in a human, comprising administering to the subject an effective dosage of a composition comprising a xenogeneic form of said human CASB933 as described herein.
  • a preferred embodiment is a method of inducing an immune response against human CASB933 using the xenogeneic CASB933 isolated from mouse, rat, pig or rhesus monkey.
  • Another preferred method of inducing an immune response according to the present invention is using an antigen composition including a live viral expression system which expresses said xenogeneic antigen.
  • the isolated xenogeneic CASB933 polypeptide will generally share substantial sequence similarity, and 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:3 over the entire length of SEQ ID NO:3. Accordingly the xenogeneic polypeptide will comprise an immunogenic fragment of the polypeptide of SEQ ID NO: 3 in which the immunogenic activity of the immunogenic fragment is substantially the same as the polypeptide of SEQ ID NO:3.
  • xenogeneic CASB933 polypeptide can be a fragment of at least about 20 consecutive amino acids, preferably about 30, more preferably about 50, yet more preferably about 100, most preferably about 150 contiguous amino acids selected from the amino acid sequences as shown in SEQ JO NO:3. More particularly xenogeneic CASB933 fragments will retain some functional property, preferably an immunological activity, of the larger molecule set forth in SEQ ID NO: 3, and are useful in the methods described herein (e.g. in pharmaceutical, immunogenic and vaccine compositions, in diagnostics, etc.).
  • the fragments will be able to generate an immune response against the human counterpart, such as the generation of cross-reactive antibodies which react with the autologous human form of CASB933 as set forth in any of the SEQ ID NO:2 and SEQ ID NO:6.
  • the xenogeneic polypeptide of the invention may be part of a larger fusion, comprising the xenogeneic CASB933 polypeptide or fragment thereof and a heterologous protein or part of a protein acting as a fusion partner as described hereabove.
  • the present invention also provides a nucleic acid encoding the aforementioned xenogeneic proteins of the present invention and their use in medicine.
  • the xenogeneic CASB933 polynucleotide for use in pharmaceutical compositions has the sequence set forth in SEQ ID N:4.
  • CASB933 polynucleotides according to the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention.
  • the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID N:4, for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%o or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters).
  • the isolated xenogeneic polynucleotide of the invention will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to the amino acid sequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4; or a nucleotide sequence complementary to said isolated polynucleotide.
  • 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 lie; 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 for use in the present invention can be prepared in any suitable manner. Such 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 polynucleotide for use according to the present invention can be inserted in a suitable plasmid or recombinant microrganism vector and used for immunisation ( 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 present invention relates to uses for polynucleotides that encode CASB933 polypeptides, particularly polynucleotides that encode the polypeptide herein designated CASB933, for use in or in preparation of the immunogenic compositions described herein.
  • the nucleotide sequence of SEQ ID NO:l is a cDNA sequence which comprises a polypeptide encoding sequence (nucleotide 122 to 1234) encoding a polypeptide of 371 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: 1 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.
  • SEQ ID NO:5 is a cDNA sequence which is an alternative isoform of CASB933 SEQ ID NO:l.
  • SEQ ID NO:5 comprises a polypeptide encoding sequence (nucleotide 120 to 1163) encoding a polypeptide of 348 aminoacids, the polypeptide of SEQ ID NO:6.
  • SEQ JJD NO:6 polypeptide sequence is an alternative form of CASB933 SEQ ID NO:2 polypeptide.
  • the nucleotide sequence encoding the polypeptide of SEQ ID NO:6 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:5 or it may be a sequence other than the one contained in SEQ ID NO:5, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:6.
  • the polypeptide of the SEQ ID NO:6 is known as Homo Sapiens ESE-la (GenPept accession number AAB65824).
  • SEQ ID NO: 7 is a cDNA sequence which is an another alternative isoform of CASB933 SEQ ID NO: 1.
  • Preferred polypeptides and polynucleotides for use according to 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 and SEQ ID NO:5 and SEQ ID NO:7 or SEQ ID NO:2 and SEQ ID NO:6, as appropriate.
  • a polynucleotide of the invention encoding CASB933 may be obtained using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human lung tumours, (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 present invention provides uses for an isolated polynucleotide comprising or consisting of:
  • 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.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 and SEQ ID NO:5 and SEQ JJJ> NO:7 may be used as hybridisation 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 and SEQ ID NO:5 and SEQ TD NO:7.
  • PCR nucleic acid amplification
  • 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. Preferred sequences in this respect are set out in SEQ ID NO:3 or SEQ ID NO:4.
  • a polynucleotide encoding a polypeptide for use in the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridisation conditions with a labeled probe having the sequence of SEQ ID NO:l and SEQ ID NO:5 and SEQ ID NO:7 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • hybridisation techniques are well known to the skilled artisan.
  • Preferred stringent hybridisation conditions include overnight incubation at a temperature in the range of 45°C - 65°C, typically 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 O.lx SSC at about 65°C.
  • SDS is present at a concentration in the range of 0.1 - 1%.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.
  • Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
  • the present invention also includes polynucleotides obtainable by screening an appropriate library under stingent hybridisation conditions with a labeled probe having the sequence of SEQ ID NO: 1 and SEQ ID NO:5 and SEQ ID NO:7 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.
  • PCR Nucleic acid amplification
  • 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.
  • the invention also provides uses for polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-tenninal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids maybe processed away from the mature protein by cellular enzymes.
  • a precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • 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.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold
  • 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 arx ⁇ Aspergillus cells
  • insect cells such as Drosophil ⁇ S2 and Spodopter ⁇ Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells.
  • 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 ⁇ l, 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.
  • polynucleotides encoding immunogenic polypeptides of the present invention are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems.
  • retroviruses provide a convenient and effective platform for gene delivery systems.
  • a selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject.
  • retroviral systems have been described (e.g., U.S. Pat. No.
  • adenoviruses persist exfrachromosomally thus minimizing the risks associated with insertional mutagenesis
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol.
  • Additional viral vectors useful for delivering the nucleic acid molecules encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome.
  • the resulting TK.sup.(-) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • avipoxviruses such as the fowlpox and canarypox viruses
  • canarypox viruses can also be used to deliver the coding sequences of interest.
  • Recombinant avipox viruses expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species.
  • the use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
  • alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in U.S. Patent Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694.
  • Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Patent Nos. 5,505,947 and 5,643,576.
  • molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention.
  • the recombinant live microorganisms described above can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
  • a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed.
  • a polynucleotide is administered/delivered as "naked" DNA, for example as described in Ulmer et al. Science 259:1145-1149, 1993 and reviewed by Cohen, Science 259:1691-1691, 1993.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
  • a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described.
  • gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject
  • Vaccines Inc. (Madison, WI), some examples of which are described in U.S. Patent Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.
  • This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.
  • compositions of the present invention include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in U.S. Patent Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.
  • 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, more particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesotheliomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or IIB of Mountain's staging) NSCLC, and related
  • 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 immunogenic/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 immunogenic 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 immunisation 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 pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or antigen presenting cell (APC) compositions of this invention.
  • An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen.
  • One prefened type of immunostimulant comprises an adjuvant.
  • Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (Glaxo SmithKline, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
  • Freund's Incomplete Adjuvant and Complete Adjuvant Difco Laboratories, Detroit, MI
  • Merck Adjuvant 65 Merck and Company, Inc., Rahway, NJ
  • AS-2 Gaxo SmithKline, Philadelphia, PA
  • aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate
  • Cytokines such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
  • the adjuvant composition is preferably one that induces an immune response predominantly of the Thl type.
  • High levels of Thl-type cytokmes e.g., IFN- ⁇ , TNF ⁇ , IL-2 and IL-12
  • Th2- type cytokines e.g., IL-4, IL-5, IL-6 and IL-10 tend to favor the induction of humoral immune responses.
  • a patient will support an immune response that includes Thl- and Th2-type responses.
  • Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffrnan, Ann. Rev. Immunol. 7:145-173, 1989.
  • Certain preferred adjuvants for eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt.
  • MPL ® adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thl response.
  • oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al. Science 273:351, 1996.
  • Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals fric, Framingham, MA); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins .
  • Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, ⁇ - escin, or digitonin.
  • the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc.
  • vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc.
  • the saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs.
  • the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM.
  • the saponins may also be formulated with excipients such as Carbopol R to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
  • the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL ® adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • a monophosphoryl lipid A and a saponin derivative such as the combination of QS21 and 3D-MPL ® adjuvant, as described in WO 94/00153
  • a less reactogenic composition where the QS21 is quenched with cholesterol as described in WO 96/33739.
  • Other preferred formulations comprise an oil-in- water emulsion and tocopherol.
  • Another particularly preferred adjuvant formulation employing QS21, 3D-MPL ® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in WO 00/09159.
  • the formulation additionally comprises an oil in water emulsion and tocopherol.
  • Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from GlaxoSmithKline, Rixensart, Belgium), Detox (Enhanzyn ® ) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. Patent Application Serial Nos.
  • n 1-50
  • A is a bond or -C(O)-
  • R is C 1-50 alkyl or Phenyl C 1-5 o alkyl.
  • One embodiment of the present invention consists of an immunogenic formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C 1-50 , preferably C -C 20 alkyl and most preferably C 12 alkyl, and A is a bond.
  • concentration of the polyoxyethylene ethers should be in the range 0.1 -20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%.
  • Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35- lauryl ether, and polyoxyethylene-23-lauryl ether.
  • Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.
  • polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant.
  • a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.
  • a carrier is also present in the immunogenic 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 immunogenic 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 immunogenic 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 immunogenic compositions 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 immunogenic composition
  • a polyvalent immunogenic composition comprising a formulation of the invention in combination with other antigens, in particular antigens useful for treating cancer and autoimmune diseases, more particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or IIB of Mountain's staging) NSCLC.
  • Such a polyvalent immunogenic composition may include a TH-1 inducing adjuvant as hereinbefore described.
  • an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs.
  • APCs antigen presenting cells
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumour effects er se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype), for instance in a preneoplasic lesions of lung cancer or lung cancer context, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesion tumours and early stage (classification stages 0, or IA, or IB, or UA, or UB of Mountain's staging) NSCLC.
  • APCs may generally be isolated from any of a variety of biological fluids and
  • Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:145-151, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antirumour immunity (see Timmerman and Levy, Ann. Rev. Med. 50:501-519, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumour-infiltrating cells, peritumoural tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL- 4, IL-13 and/or TNF ⁇ to cultures of monocytes harvested from peripheral blood.
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF ⁇ , CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • Dendritic cells are conveniently categorized as ''immature'' and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes.
  • Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc ⁇ receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4- IBB).
  • APCs may generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein.
  • a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al.
  • Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumour polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
  • the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule).
  • a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
  • compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.
  • Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable.
  • the formulation preferably provides a relatively constant level of active component release. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired.
  • the formulation of such compositions is well within the level of ordinary skill in the art using known techniques.
  • Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like.
  • illustrative delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Patent No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638).
  • a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • biodegradable microspheres e.g., polylactate polyglycolate
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.
  • Modified hepatitis B core protein carrier systems such as described in WO/99 40934, and references cited therein, will also be useful for many applications.
  • Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Patent No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.
  • compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., gly
  • compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use.
  • formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
  • a pharmaceutical composition may be stored in a freeze- dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • compositions disclosed herein may be delivered via oral administration to an animal.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al, Nature 1997 Mar 27;386(6623):410-4; Hwang et al, Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U. S. Patent 5,641,515; U. S. Patent 5,580,579 and U. S. Patent 5,792,451).
  • Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose
  • Narious other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar, or both.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • these formulations will contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
  • Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U. S. Patent 5,466,468).
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution for parenteral administration in an aqueous solution, should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
  • compositions disclosed herein may be formulated in a neutral or salt form.
  • Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • solvents dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212.
  • the delivery of drugs using intranasal microparticle resins (Takenaga et al, J Controlled Release 1998 Mar 2;52(l-2):81-7) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871) are also well-known in the pharmaceutical arts.
  • illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045.
  • compositions of the present invention are used for the introduction of the compositions of the present invention into suitable host cells/organisms.
  • the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.
  • liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol 1998 Jul; 16(7): 307-21; Takakura, Nippon Rinsho 1998 Mar;56(3):691-5; Chandran et al, Indian J Exp Biol. 1997 Aug;35(8):801-9; Margalit, Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Patent 5,567,434; U.S. Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each specifically incorporated herein by reference in its entirety).
  • Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al, J Biol Chem. 1990 Sep 25 ;265(27): 16337-42; Muller et al, DNA Cell Biol. 1990 Apr;9(3):221-9).
  • liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.
  • liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLNs).
  • MNs multilamellar vesicles
  • the invention provides for pharmaceutically- acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al, Drug Dev hid Pharm. 1998 Dec;24(12):l 113-28).
  • such ulfrafine particles may be designed using polymers able to be degraded in vivo.
  • Such particles can be made as described, for example, by Couvreur et al, Crit Rev Ther Drug Carrier Syst. 1988;5(l):l-20; zur Muhlen et al, Eur J Pharm Biopharm. 1998 Mar;45(2): 149-55; Zambaux et al. J Controlled Release. 1998 Jan 2;50(l-3):31-40; and U. S. Patent 5,145,684.
  • 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.
  • Surrogate tumour markers can be used to diagnose different forms and states of cancer, more particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or JJB of Mountain's staging) NSCLC.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung 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 cancer and autoimmune disease, more particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or IIB of Mountain's staging) NSCLC, 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.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung cancer
  • 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 hybridisation 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.
  • mRNA corresponding to SEQ ID NO:l and SEQ ID NO:5 and SEQ ID NO:7 relative to normal level is indicative of the presence of cancer in the patient, more particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or UB of Mountain's staging) NSCLC.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung cancer
  • 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.
  • 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 hybridisation analysis which relies on the specificity of nucleotide interactions.
  • SAGE serial analysis of gene expression
  • differential display methodology For example, US 5,776,683
  • hybridisation 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 NO:l and SEQ ID NO:5 and SEQ ID NO:7.
  • 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.
  • a large number of 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.
  • arrays of the polynucleotide sequences SEQ ID NO:l and SEQ ID NO:5 and SEQ ID NO:7 can be used to determine if any of the polynucleotides are differentially expressed between a normal and cancer cell, more particularly preneoplasic lesions of lung cancer and lung cancer cell, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or
  • an array of oligonucleotides probes comprising the SEQ ID NO: 1 and SEQ ID NO:5 and SEQ ID NO:7 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al. Science, Vol 274, pp 610-613 (1996)).
  • 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:
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 and SEQ ID NO:6, 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, Mendehan Inheritance in Man (available on-hne 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 maybe 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 et al, Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).
  • the above-described antibodies maybe 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, more particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or UB of Mountain's staging) NSCLC, autoimmune disease and related conditions.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung cancer
  • squamous carcinoma epidermoid carcinoma
  • adenocarcinoma including bronchoalveolar
  • large cell (undifferentiated) carcinoma or carcinoids
  • 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 diseasesmore particularly preneoplasic lesions of lung cancer and lung cancer, for instance small cell lung cancer (SCLC), or non small cell lung cancer (NSCLC) such as squamous (epidermoid) carcinoma, adenocarcinoma (including bronchoalveolar) and large cell (undifferentiated) carcinoma, or carcinoids, or bronchial gland tumours or mesothehomas, even more particularly NSCLC preneoplasic lesions and early stage (classification stages 0, or IA, or IB, or UA, or UB of Mountain's staging) NSCLC, related to either a presence of, an excess of, or an under- expression of, CASB933 polypeptide activity.
  • SCLC small cell lung cancer
  • NSCLC non small cell lung cancer
  • the present invention further provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the CASB933 polypeptide.
  • agonists or antagonists maybe 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:
  • 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:
  • a 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: (a) determining in the first instance the three-dimensional structure of the polypeptide;
  • Gene therapy may also be employed to effect the endogenous production of CASB933 polypeptide by the relevant cells in the subject.
  • 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 Fullerton, 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.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W, ed.
  • 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. 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S, et al, NCBI NLM NM Befhesda, 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:
  • a program useful with these parameters 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 and SEQ ID NO:5 and SEQ TD NO:7, 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: 1 and SEQ ID NO:5 and SEQ TD NO:7 by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ LD NO:l and SEQ ID NO:5 and SEQ ID NO:7, 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 SEQ ID NO:5 and SEQ TD NO:7, 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
  • SEQ ID NO:2 and SEQ ID NO:6 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 maybe identical to the reference sequence of SEQ ID NO:2 and SEQ TD NO:6, 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 and SEQ ID NO:6 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 and SEQ TD NO:6, or: n a ⁇ x a - (x 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 SEQ ID NO:6, 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.
  • Example 1 Subtractive cDNA cloning of tumour-associated antigen (TAA) candidates.
  • RNA is extracted from frozen (-70°C) preneoplasic biposies of patients suffering from non small cell lung carcinoma (NSCLC) and normal samples using the TriPure reagent and protocol (Roche Applied Science).
  • Target RNA is prepared by pooling total RNA from 30 or 10 preneoplasic tumour samples.
  • Driver RNA is prepared by pooling total RNA from 12 normal tissues (namely adrenal gland, brain, kidney, livern skeletal muscle, pancreas, spinal cord, spleen, colon, heart, bone marrow, lymph node).
  • Messenger RNA is purified from total RNA using oligo-dT magnetic bead technology (Dynal) and quantified by spectrofluorimetry (BioRad).
  • Target and driver mRNA are reverse transcribed into cDNA using one of the following three strategies : 1) Target sequences for PCR oligonucleotides are introduced onto the ends of the newly synthesised cDNA during reverse transcription using the template switching capability of reverse transcriptase (ClonTech SMART PCR cDNA synthesis kit); 2) Alternatively, the target and driver mRNA are reverse transcribed into cDNA using an oligo-dT primer and converted to double-strand cDNA; the cDNA is cleaved with Rsal and linkers for PCR amplification are ligated onto the extremities of the cDNA fragments; 3) Target sequences for PCR amplifcation are introduced onto the ends of mRNA using GeneRacer technology (Invitrogen Corp.).
  • target and driver cDNA are amplified by long range PCR (ClonTech SMART PCR Synthesis Kit and Advantage PCR Polymerase Mix) and used as starting material for subtractive cloning.
  • long range PCR ClonTech SMART PCR Synthesis Kit and Advantage PCR Polymerase Mix
  • cycling conditions and optimisation of the number of PCR cycles are as described in the Advantage PCR protocol.
  • Example 2 Differential Screening of cDNA arrays.
  • Identification of tumour-associated genes in the subtracted cDNA library is accomplished by differential screening : Total bacterial DNA is extracted from 100 ⁇ l over-night cultures. Bacteria are lysed with guanidium isothiocyantate and the bacterial DNA is affinity purified using magnetic glass (Boehringer). Plasmid inserts are recovered from the bacterial DNA by Advantage PCR amplification (Clontech). The PCR products are dotted onto two nylon membranes to produce high-density cDNA arrays using the Biomek 96 HDRT tool (Beekman). The spotted cDNA is covalently linked to the membrane by UN irradiation. The first membrane is hybridised with a mixed cD ⁇ A probe prepared from the tumour of a single patient.
  • the second membrane is hybridised with an equivalent amount of mixed cD ⁇ A probe prepared from corresponding normal tissue from the cancerous organ of the same patient.
  • the probe cD ⁇ A is prepared by PCR amplification as described above and is labelled using the AlkPhos Direct System (Amersham). Hybridisation conditions and stringency washes are as described in the AlkPhos Direct kit. Hybridised probe is detected by chemilumrnescence. Hybridisation intensities for each cD ⁇ A fragment on both blots are measured by film densitometry or direct measurement (BioRad Fluor-S Max). The ratio of the tumour to normal hybridisation intensities (TIN) is calculated for each gene to evaluate the degree of over-expression in the tumour.
  • Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) is used to evaluate and compare mR ⁇ A transcript abundance of the candidate antigen in panels of tumour and preneoplasic tissues of lung from multiple patient samples, of normal tissues, of different normal and tumour cell lines of lung, and finally of lung tumours grown in vivo in SCUD mice. This analysis is important to establish the lung tumour specificity of CASB933 expression, which is an important criterion a good antigen candidate must fulfil.
  • RNA is extracted using TriPure reagent (Boehringer) and DNAse treatment from snap frozen biopsies of preneoplasic lesions of lung, early stage (stages lb or Ua or 1Tb) non small cell lung carcinoma (NSCLC), late stage (stages Ilia or Ulb or IV) NSCLC, and several normal tissues, or from normal and tumour cell lines or lung tumours grown in vivo in SCID mice. Lung cancer stages are determined following the Mountain's classification (Mountain CF. Revisions in the international system for satging lung cancer. Chest 1997, 111:1710-1717).
  • RNA Quantification of total RNA is performed by spectrofluorimetry (VersaFluor, BioRad) using Sybrll dye (Molecular Probes), and cDNA is obtained from the quantified total RNA by reverse transcription using a poly-dT oligonucleotide and Expand (Roche) reverse transcriptase.
  • Real-time PCR reactions are assembled according to standard PCR protocols using 50 ng of totalRNA for each reaction, and performed using either SYBR green PCR core kit or TaqMan PCR core kit (Applied Biosystems) following the manufacturer instructions. 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. Several Ct values are obtained for each samples : a CtXY for each sample XY is calculated on CASB933 transcript. An another Ct (CtA) is also obtained on the ⁇ -Actin gene in parallel, as an internal reference, for all samples.
  • CtA CtA
  • 1 ' tXY is an estimate of the relative TAA transcript level of the sample, standardised with respect to Actin transcript level. A value of 1 thus suggests the candidate antigen and Actin have the same expression level.
  • Absolute levels of CASB933 transcript, as assessed by SYBR green quantitative RT-PCR technology, in preneoplasic lesions of lung, early and late stage lung cancers and normal tissues is shown in figures 1 to 4.
  • Comparison of CASB933 transcript abundance in preneoplasic lesions of lung, early and late stage lung cancers to normal tissues is depicted in table 1. It clearly show CASB933 transcript is over-expressed in most of patients suffering from lung preneoplasic lesions and early stage lung cancers. Overexpression fold in early stage lung cancer population of patients ranges from 4 to 8. These result were further confirmed on early stage lung cancer patient population by the TaqMan quantitative RT-PCR technology, adding more specificity to the assay by using a CASB933 specific probe.
  • TaqMan results are shown in figures 5 and 6 and table 2. Taken together, these results suggest CASB933 transcript in over-expressed between 4 to 8 times in a majority of patients suffering from preneoplasic lesions of lung cancer and early stage lung cancer (stages IB and UA and IIB). CASB933 lung preneoplasic lesion and lung tumour specificity agrees with its use as a diagnostic and vaccinating agent in a lung cancer context.
  • Table 1 CASB933 transcript overexpression coverages and levels in lung cancer patient populations as compared to normal tissues (quantitative RT-PCR, SYBR green technology).
  • Table 2 CASB933 transcript overexpression coverages and levels in an early stage lung cancer population as compared to normal tissues (quantitative RT-PCR, TaqMan technology).
  • Example 4 Real time PCR analysis of CASB933 on preneoplasic lesions of lung and normal cDNA libraries.
  • cDNA libraries from lung preneoplasic lesions and normal tissues mRNA was purified from a pool of 30 preneoplasic biopsies (endoscopic sampling) and from a pool of normal tissues.Then the same protocol was applied to both mRNA preparations :350 ng of mRNA were reverse-transcribed using Superscript U enzyme (Life Technologies). The second cDNA strand was synthesized using RNAseH-DNA polymerase I and was blunted with the Pfu DNA polymerase. T3 promoter was specifically ligated at the cDNA 5' end. cDNA was attached to streptavidin beads (Dynal) via its biotinylated 3 ' end. In vitro transcribed mRNA (using T3 RNA polymerase) was purified on oligo-d(T) magnetic beads (Dynal). cDNA library was constructed from 5 ⁇ g of purified RNA using the classical Stratagene protocol.
  • Example 5 DNA microarrays.
  • DNA micro-arrays are used to examine mRNA 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 tumours and normal tissues.
  • Examples of current technologies for production of DNA micro-anays include 1) The Affymetrix "GeneChip" arrays in which oligonucleotides are synthesised on the surface of the chip by solid phase chemical synthesis using a photolithographic process 2) DNA spotting technology in which small volumes of a DNA solution are robotically deposited and then immobilised onto the surface of a solid phase (e.g. glass).
  • the chips are hybridised with cDNA or cRNA that has been extracted from the tissue of interest (e.g. normal tissue, tumour etc...) and labelled with radioactivity or with a fluorescent reporter molecule.
  • the labelled material is hybridised to the chip and the amount of probe bound to each sequence on the chip is determined using a specialised scanner.
  • the experiment can be set-up with a single fluorescent reporter (or radioactivity) or, alternatively, can be performed using two fluorescent reporters.
  • each of the two samples is labelled with one of the reporter molecules.
  • the two-labelled samples are then hybridised competitively to the sequences on the DNA 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 "DNA Microarrays: A practical approach. Schena M.
  • Example 6 Northern-Southern blot analysis.
  • 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).
  • Example 8 In silico detection of the full length cDNA sequence.
  • EST sequence databases are screened with experimentally obtained cDNA sequence fragments, using the Blast algorithm (Altschul, S.F, Gish, W, Miller, W, Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol. 215:403-41). The aim is to search for overlapping or longer identical EST sequences. Matched EST sequences are then assembled together, using the SeqMan software of the Lasergene package (DNASTAR). The consensus sequence of the resulting assembly is an EST- derived longer cDNA. This EST-derived cDNA is further analysed using the GeneMark software to find a potential open reading frame (ORF).
  • Blast algorithm Altschul, S.F, Gish, W, Miller, W, Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol. 215:403-41). The aim is to search for overlapping or longer identical EST sequences.
  • the translated sequence of the ORF is compared with protein databases, using the Blast algorithm, to find homologues. If any, the homologous protein sequences are further used to complete the cDNA prediction by searching for genomic contig homologies using the Wise2 algorithm, leading to a genome-derived, virtual cDNA sequence.
  • This virtual cDNA in finally assembled with EST-derived cDNA, and the new consensus cDNA undergoes a final check against ESTs to confirm the Wise2 prediction, and conect potential sequencing enors and frameshifts.
  • the virtual cDNA is considered as a virtual full length cDNA once a full ORF (from start to stop codons), with clear protein homologies and coding potential. Virtual full-length cDNA sequence is experimentally checked as described in Example 10.
  • Example 9 PCR analysis in CASB933 in other tumours.
  • Presence of CASB933 transcript in tumours was assessed by PCR on two cDNA library made from tumours (see below, construction ofcDNA libraries from tumours). PCR reactions were assembled and ran according to standard conditions, using 10 ng of library plasmid as template.
  • RNA is extracted from solid tumours (Tri-Pure, Roche). mRNA is purified on oligo-d(T) magnetic beads (Dynal). 5 ⁇ g of poly A+ mRNA was reverse-transcribed using Superscript II polymerase. Second strand was synthesised using RNAseH-DNA polymerase I and blunted with the Pfu DNA polymerase. EcoRI adapters were specifically ligated at the cDNA 5' end. cDNA was size-fractionated using a Sepharose CL-2B column and ligated into phage arms (Uni-ZAP lambda, Sfratagene). Phage DNA was in vitro packaged and amplified in E. coli. cDNA inserts were transfened into plasmids using the mass excision protocol (Sfratagene).
  • PCR amplification ofcDNA from preneoplasic lesions mRNA was extracted and purified from a pool of 10 preneoplasic biopsies (endoscopic sampling).
  • cDNA was synthesised using the GeneRacer technology (Invitrogen) which ensures the amplification of only full-length transcripts. 250 ng of mRNA were treated with CIP. mRNA 5' ends were decapped with TAP (Tobacco Acid Pyrophosphatase) and were ligated to a specific RNA oligonucleotide. The ligated mRNA was reverse transcribed into cDNA using an oligod(T) tailed primer. Amplification of cDNA was performed using both GeneRacer flanking primers (Advantage, Clontech).
  • Example 10 Experimental Identification of the full length cDNA sequence. To experimentally confirm CASB933 virtual full length sequence, transcript amplification product with selected primers on a tumour cDNA library containing the transcript is diluted and re-ran on an agarose gel to precisely check product size.
  • tumour cDNA library An alternative approach is a screening of a tumour cDNA 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 phages 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 M13 bacteriophage by in vivo excision. The bacteriophages are 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.
  • ESTs Expressed Sequence Tags
  • Such database currently provides a massive amount of human ESTs (2 10 6 ) from several hundreds ofcDNA tissue libraries, including tumour tissues from various types and states of disease.
  • Blast informatics tools
  • a comparison search of the CASB933 sequence is performed in order to have further insight into tissue expression.
  • the following table 3 describes CASB933 EST number distribution by tissue type :
  • Recombinant proteins may be expressed in two microbial hosts, E. coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris). 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 (fFP).
  • an affinity fusion partner (AFP) useful for facilitating further purification is included at the C-terminal end.
  • the recombinant product is characterised by the evaluation of the level of expression and the prediction of further solubility of the protein by analysis of the behaviour in the crude extract.
  • the recombinant proteins are visualised in stained gels and identified by Western blot analysis using specific antibodies.
  • 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-H-NTA from Qiagen) that will specifically retain the recombinant protein.
  • IMAC Ion Metal Affinity Chromatography
  • 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).
  • Rabbits are immunised , intramuscularly (I.M.) , 3 times at 3 weeks intervals with lOO ⁇ g of protein, formulated in the adjuvant 3D-MPL/QS21. Three weeks after each immunisation a blood sample is taken and the antibody titer estimated in the serum by ELISA using the protein as coating antigen following a standard protocol.
  • 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.
  • Anti-peptide antibodies Besides being highly tumour specific, the second critical criterion for a candidate vaccine evaluation is its immunogenicity. One way of assessing the immunogenicity of a protein is to immunise na ⁇ ve animals with synthetic peptides derived from the antigen sequence, and which reproduce natural epitopes. The generated anti-peptide antibodies will then tend to recognise the native antigen, therefore showing a specific immune response can be raised against the candidate vaccine antigen.
  • One or several peptides are selected from CASB933 antigen sequence for their immunogenic potential.
  • Two rabbits are immunised with each peptide.
  • the selected peptides were conjugated to a carrier protein (KLH).
  • Rabbits were intramuscularly immunised with CASB933 peptides, 3 times at 3 to 4 weeks intervals with 200 ⁇ g of conjugate formulated with Freund's adjuvant.
  • PP second immunisation
  • GP third immunisation
  • blood samples were taken, and the CASB933 specific antibody titers were estimated in the serum by ELISA.
  • a dose range of 0, 200 and 400 ⁇ g of the antigen conjugate is reproduced.
  • ELIS ELIS As were done in triplicate for each immunising peptide and rabbit serum, and performed as follows : 96 well microplates were coated at 4°C during 16 hours with either 100 ng of CASB933 peptide or 100 ng of KLH as coating antigens. After 2-hour saturation at 25°C with BSA (1 mg/ml), serial dilution of the rabbit sera was added for 2 hours at 25°C (starting at dilution 1/100). Anti rabbit antibody, conjugated with universal- HRP, was then added at dilution 1/1000 for 2 hours at 25°C as secondary antibody. Plates were washed and OPD (0.4 mg/ml) is added for 30 min at 25°C. Reaction was stopped with H2S04 4M, and OD measured at 492 nm.
  • Spleen cells are fused with the SP2/0 myeloma according to a standard protocol using
  • 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.
  • the immunological relevance of the antigen of the invention can be assessed by in vitro priming of human T cells. All T cell lymphocyte lines and dendritic cells are derived from PBMCs (peripheral blood mononuclear cells) of healthy donors (preferred HLA-A2 subtype). An HLA-A2.1/Kb transgenic mouse model 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 ⁇ -IFN production of the CD8+ lines in response to the antigen or antigen derived-peptides is tested using standard procedures.
  • transgenic mice are immunised 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 analysed in the human system.
  • Human dendritic cells cultured according to Romani N, Gruner S, Brang D, Kampgen E, Lenz A,rbacher B, Konwalinka G, Fritsch PO, Steinman RM, Schuler G. (1994) Proliferating dendritic cell progenitors in human blood. J Exp Med. 180:83-93.
  • 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). Whole gene-based approach
  • 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 C J, Prevette T, Cormier J, Overwijk W, Roden M, Restifo NP, Rosenberg S A, Marincola FM. (1997) Dendritic cells infected with poxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro.
  • CD 8+ lines are tested on cDNA-transfected tumour cells as indicated above. Peptide specificity and identity is determined to confirm the immunological validation.
  • CD4+ T-cell immune response can also be assessed.
  • Generation of specific CD4+ T-cells is made using dendritic cells loaded with recombinant purified protein or peptides to stimulate the T-cells.
  • HLA Class I binding peptide sequences were predicted either by the Parker's algorithm (Parker K, Bednarek M, Coligan J (1994). Scheme for ranking potential HLA-
  • HLA-A2.1/Kb transgenic mice model The HLA Class ⁇ binding peptide sequences are predicted using the Tepitope algorithm, with a score cut-offset to 5 (Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J. (1999) Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat Biotechnol. 17: 555-61).

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Abstract

Utilisation des polypeptides et polynucléotides CASB933 en diagnostique, compositions et vaccins immunogènes utilisés dans la prévention et le traitement thérapeutique des cancers et notamment des lésions prénéoplasiques provoquées par le cancer du poumon, notamment le cancer pulmonaire à petites cellules (SCLC) ou un cancer pulmonaire 'non à petites cellules' (NSCLC) tel que l'éphithélioma spinocellulaire (épidermoïde), l'adénocarcinome (y compris l'adénocarcinome broncho-alvéolaire) et le carcinome à grandes cellules (non différentié), ou des carcinoïdes, ou des tumeurs bronchiales ou des mésothéliomes et plus particulièrement des lésions prénéoplasiques NSCLC à un stade précoce (stades de classification 0, ou IA, ou IB, ou IIA, ou IIB selon la classification des stades de Mountains).
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