EP2012823A2 - Produit de thérapie combinée et utilisations de celui-ci - Google Patents

Produit de thérapie combinée et utilisations de celui-ci

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Publication number
EP2012823A2
EP2012823A2 EP07732700A EP07732700A EP2012823A2 EP 2012823 A2 EP2012823 A2 EP 2012823A2 EP 07732700 A EP07732700 A EP 07732700A EP 07732700 A EP07732700 A EP 07732700A EP 2012823 A2 EP2012823 A2 EP 2012823A2
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EP
European Patent Office
Prior art keywords
combination product
agent
cells
product according
hcap18
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP07732700A
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German (de)
English (en)
Inventor
Mona STÅHLE
Günther WEBBER
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Lipopeptide AB
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Lipopeptide AB
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Publication of EP2012823A2 publication Critical patent/EP2012823A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a new combination of pharmaceutically-active agents for use in the treatment of cancer.
  • the invention provides a combination therapy capable of inhibiting the proliferation and/or metastasis of breast cancer cells.
  • Antimicrobial proteins are key effectors in the innate immune system.
  • Human cathelicidin antimicrobial protein hCAP18 the only known cathelicidin in humans, consists of a conserved cathelin domain and a variable C4erminus, called LL-37 (Gudmundsson et al, 1996, Eur J Biochem 1238:325-32; Zanetti et al, 1995, FEBS Lett 374:1-5).
  • Extracellular proteolytic processing of the holoprotein releases the LL-37 peptide, which has broad antimicrobial activity (Gudmundsson et al, 1995, Proc Natl Acad Sd USA 92:7085-9; Agerberth et al, 1995, Proc Natl Acad Sd USA 92:195-99) as well as effects on host cells, some of which are mediated by the G-protein-coupled receptor, formyl peptide receptor-like 1 (FPRLl) (Yang et al, 2000, J Exp Med 192:1069-74; Koczulla et al, 2003, J Clin Invest 111:1665-72).
  • FPRLl formyl peptide receptor-like 1
  • hCAPl ⁇ is present in leucocytes (Cowland et al, 1995, FEBS Lett 368:173-76) and is expressed in skin and other epithelia where it is upregulated in association with inflammation (Cowland et al, 1995, FEBS Lett 368:173-76; Vxohm et al, 1997, J Biol Chem 272:15258-63) and injury (Dorschner et al, 2001, J Invest Dermatol 117:91-97; Heilborn et al, 2003, J Invest Dermatol 120:379-89) consistent with a role in innate barrier protection.
  • the epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four receptors, EGF-R (ErbBl), ErbB2 (Neu), ErbB3, and ErbB4, Members of the EGF-R family contain a cytoplasmic tyrosine kinase domain, a single transmembrane domain, and an extracellular domain that is involved in ligand binding and receptor dimerization. Activation of the EGF-R results in the initiation of a diverse array of cellular pathways.
  • EGF-R In response to toxic environmental stimuli, such as ultraviolet irradiation, or to receptor occupation by EGF, the EGF-R forms homo- or heterodimers with other family members. Each dimeric receptor complex will initiate a distinct signalling pathway by recruiting different Src homology 2 (SH2)-containi ⁇ g effector proteins. Dimerization results in autophosphorylation. initiating a downstream cascade of events culminating in cellular responses such as cell proliferation or apoptosis.
  • SH2 Src homology 2
  • Grb guanine nucleotide releasing factor
  • the Grb-SOS complex can either bind directly to phosphotyrosine sites in the receptor or indirectly through She. These protein interactions bring SOS in close proximity to Ras, allowing for Ras activation This subsequently activates the ERK and JNK signalling pathways that, in turn, activate transcription factors, such as c-fos, AP-I, and EIk-I, that promote gene expression and contribute to cell proliferation.
  • a combination product comprising:
  • each of components (A) and (B) is formulated in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • an 'agent' we include all chemical entities, for example oligonucleotides, polynucleotide, polypeptides, peptidomimetics and small compounds.
  • the combination product of the invention comprises a pharmaceutical formulation including a first agent that inhibits the biological activity of hCAP18/LL-37, a second agent that inhibits the biological activity of an EGF receptor, and a pharmaceutically-acceptable adjuvant, diluent or carrier.
  • (B) a pharmaceutical formulation including a second agent that inhibits the activity of EGF receptors, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (A) and (B) are each provided in a form that is suitable for administration in conjunction with the other.
  • components (A) and (B) of the kit of parts may be:
  • the term "administration in conjunction with” includes that the two components of the combination product (the first agent that inhibits activity of hCAP18/LL-37 and the second agent that inhibits the activity of EGF receptors) are administered (optionally repeatedly), either together, or sufficiently closely in time, to enable a beneficial effect for the patient, that is greater, over the course of the treatment of the relevant condition, than if either a formulation comprising the first agent that inhibits activity of hCAP18/LL-37, or a formulation comprising the second agent that inhibits the activity of EGF receptors, are administered (optionally repeatedly) alone, in the absence of the other component, over the same course of treatment. Determination of whether a combination provides a greater beneficial effect in respect of, and over the course of treatment of, a particular condition will depend upon the condition to be treated or prevented, but may be achieved routinely by the skilled person.
  • the EGF receptor being inhibited is at least one selected from ErbBl (EGF-R), ErbB2, ErbB3 and ErbB4.
  • the first agent inhibits the biological activity of hCAP18/LL-37 by altering the transcription, translation, cleavage and/or binding properties of hCAP18/LL-37 and/or the second agent inhibits the biological activity of an EGF receptor by altering the transcription, translation and/or binding properties of an EGF receptor.
  • Such agents may be identified using methods well known in the art, such as:
  • the first agent is an inhibitor of the transcription of hCAP18/LL-37 and/or file second agent is an inhibitor of the transcription of an EGF receptor.
  • the first agent is an inhibitor of the translation of hCAP18/LL-37 and/or the second agent is an inhibitor of the translation of an EGF receptor.
  • the first agent is an inhibitor of the binding properties of hCAP18/LL-37 and/or the second agent is an inhibitor of the binding properties of an EGF receptor.
  • the agent(s) may alter the conformation of hCAP18/LL-37 such that it is no longer able to bind to its receptor.
  • the first agent is an hCAP18/LL-37 receptor antagonist and/or the second agent is an EGF receptor antagonist. It will be appreciated by persons skilled in the art that the agent(s) may inhibit biological activity of by blocking receptor function directly, i.e. by acting as an receptor antagonist or indirectly.
  • the hCAP18/LL-37 receptor is a receptor of the FRP family.
  • the first agent is capable of inhibiting the biological activity of hCAP18/LL-37 and/or the second agent is capable of inhibiting the biological activity of EGF receptors.
  • the first agent may be an inhibitor of cathelicidin proteolytic processing, for example a protease inhibitor (such as proteinase 3).
  • biological activity of hCAP18/LL-37 and/or EGF receptors is inhibited in cancer cells selectively.
  • the agent inhibits the biological activity of hCAP18/LL-37 and/or EGF receptors to a greater extent than it modulates the activity of other proteins in the cancer cells.
  • the agent inhibits only the biological activity of hCAP18/LL-37 and/or EGF receptors, although it will be appreciated that the expression and activity of other proteins within the cancer cells may change as a downstream consequence of a selective inhibition of hCAP18/LL-37 and/or EGF receptors.
  • inhibition of the biological activity of hCAP18/LL-37 and/or EGF receptors by an agent of the invention may be in whole or in part.
  • the agent may inhibit the biological activity of hCAP18/LL-37 by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to the biological activity of hCAP18/LL-37 and/or EGF receptors in cancer cells which have not been exposed to the agent
  • the first agent is capable of inhibiting the biological activity of hCAP18/LL-37 by 50% or more compared to the biological activity of hCAP18/LL-37 in cancer cells which have not been exposed to the agent
  • the second agent is capable of inhibiting the biological activity of ERBB2 by 50% or more compared to the biological activity of ERBB2 in cancer cells which have not been exposed to the agent.
  • the first and/or second agent is selected from the group consisting of short interfering RNA (siRNA) molecules, antisense oligonucleotides, compounds with binding affinity for hCAP18/LL-37 and/or EGF receptors and small inhibitor compounds.
  • siRNA short interfering RNA
  • the first agent may be a small inhibitor compound such as antagonists to vitamin D, for example ZK159222 (Schering AG) and TEI-9647 (Tejin Institute for Medical Research, Tokyo), and antagonists to vitamin A, for example AGN193109 (Allergen Pharmaceuticals).
  • vitamin D for example ZK159222 (Schering AG) and TEI-9647 (Tejin Institute for Medical Research, Tokyo)
  • antagonists to vitamin A for example AGN193109 (Allergen Pharmaceuticals).
  • EGF receptor inhibitors examples include the drug Herceptin (trastuzumab, Genentech) a monoclonal antibody with specificity for ErbB2, the drug Erbitux (cetuximab, Bristol-Meyers Squibb) a monoclonal antibody with specificity for EGF-R (ErbBl)), other monoclonal antibodies such as MAB225, the small molecule IRESSA (gefitinib, Astra Zeneca) that inhibits EGF receptors by inhibiting tyrosine kinases and other tyrosine kinase inhibitors ⁇ e.g. PDl 53035, GW572016 and others, available commercially from suppliers such as Calbiochem/Merck.
  • Herceptin tacuzumab, Genentech
  • Erbitux cetuximab, Bristol-Meyers Squibb
  • MAB225 the small molecule IRESSA (gefitinib, Astra Zeneca) that inhibits EGF receptors by inhibiting
  • At least one of the first and second agents is a short interfering RNA (siRNA) molecule.
  • siRNA short interfering RNA
  • RNA interference is a two-step process.
  • the first step which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the Rnase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA) each with 2-nucleotide 3 ' overhangs (Hutvagner & Zamore, 2002, Ciirr. Opin. Genetics and Development 12:225-232; Bernstein, 2001, Nature 409:363- 366).
  • siRNA small interfering RNAs
  • the siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC.
  • the active RISC targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA (Hutvagner & Zamore, 2002, supra.; Hammond et ah, 2001, Nat. Rev. Gen. 2:110-119 (2001); Sharp, 2001, Genes. Dev. 15:485-90).
  • each RISC contains a single siRNA and an RNase (Hutvagner & Zamore, 2002, supra.).
  • RNAi RNAi RNAi RNAi amplification step within the RNAi pathway. Amplification could occur by copying of the input dsRNAs ⁇ vhich would generate more siRNAs, or by replication of the siRNAs formed. Alternatively, or additionally, amplification could be effected by multiple turnover events of the RISC (Hammond et al, 2001, supra.; Hutvagner & Zamore, 2002, supra). Additional information on RNAi can be found in the following reviews, Tuschl, 2001, Chem. Biochem. 2:239-245, Cullen, 2002, Nat. Immunol. 3:597-599 and Brantl, 2002, Biochem. Biophys Act. 1575: 15-25.
  • RNAi molecules suitable for use with the present invention can be effected as follows. First, the hCAP18/LL-37 mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites.
  • siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex (Tuschl, ChemBiochem. 2:239- 245). It will be appreciated, however, that siRNAs directed at untranslated regions may also be effective.
  • potential target sites are compared to an appropriate genomic database (e.g. human, mouse, rat, etc.) using sequence alignment software, such as the BLAST fwww.ncbi.rjIm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
  • sequence alignment software such as the BLAST fwww.ncbi.rjIm.nih.gov/BLAST/.
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the SLRNAS but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • the siRNA molecule inhibiting hCAP18/LL-37 comprises a fragment of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof.
  • HCAP18/LL-37 nxRNA (Accession No. MN 004345)
  • the siRNA molecule comprises a fragment of the nucleotide sequence transcribed from ENSG00000164047 (genomic sequence).
  • the siRNA molecule inhibiting an EGF receptor comprises a fragment of the nucleotide sequence of mRNA encoding ErbBl (EGF-R), ErbB2, ErbB3 or ErbB4, or a variant thereof.
  • mRNA sequences are as follows:
  • fragment we mean at least 10 nucleotides, for example at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • variant we mean that the nucleotide sequence shares at least 90% sequence identity with a fragment of SEQ ID NO:1 or at least 95% sequence identity with a fragment of SEQ ID NO:1, for example at least 95%, 96%, 97%, 98% or 99% sequence identity.
  • the percent sequence identity between two polynucleotides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polynucleotides whose sequences have been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (as described in Thompson et al, 1994, Nuc. Acid Res. 22:4673-4680).
  • the parameters used may be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
  • the BESTFIT program may be used to determine local sequence alignments.
  • the siRNA molecule is 19 to 23 nucleotides in length.
  • At least one of the first and second agents is an antisense oligonucleotide.
  • the design of antisense molecules which can be used to decrease efficiently hCAP18/LL-37 and/or EGF receptor levels/activity requires consideration of two aspects important to the antisense approach.
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the cancer cells, while the second aspect is design of an oligonucleotide which specifically binds the designated rnENA within cells in a way which inhibits translation thereof.
  • the prior art teaches a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types (for example, see Gut, 1998, J MoI Med 76:75-6; Kronenwett et al, 1998, Blood 91:852-62; Rajur et al, 1997, Bioconjug Chem 8:935-40; Lavigne et al, 1997, Biochem Biophys Res Commun 237:566-71; AoId et al, 1997, Biochem Biophys Res Commun 231:540-5).
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used (Hohnlund et al, 1999, Curr Opin MoI Ther 1:372-85; Gerwitz, 1999, Curr Opin MoI Ther 1:297-306). More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model (Uno et al, 2001, Cancer Res 61:7855-60).
  • the antisense oligonucleotide inhibiting hCAP18/LL-37 comprises a fragment of the nucleotide of SEQ ID NO: 1, or a variant thereof.
  • the antisense oligonucleotide inhibiting an EGF receptor comprises a fragment of the nucleotide sequence of mRNA encoding ErbBl (EGF-R), ErbB2, ErbB3 or ErbB4, or a variant thereof.
  • the antisense oligonucleotide is 15 to 35 nucleotides in length.
  • oligonucleotides For example, 20-mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor rnRNA (Witters et al, Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, JNeurosurg 91:261-7 (1999)).
  • oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases.
  • modified oligonucleotides e.g. having altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced with another linkage.
  • Agrawal et al (1988) Proc. Natl. Acad. Sd USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-I using oligonucleotide phosphoramidates and phosphorothioates.
  • Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo.
  • Shaw et al (1991) in Nucleic Acids Res. 19, 747- 750 report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.
  • oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesised and applied.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridisation and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells.
  • the present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage.
  • Linkages examples include phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates. Such examples are illustrative, rather than limiting, since other internucleotide linkages are well known in the art. The synthesis of oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.
  • Oligonucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5' or 3' terminal nucleotides.
  • a reagent for capping is commercially available as Amino-Linlc ⁇ TM from Applied BioSystems Inc, Foster City, CA. Methods for capping are described, for example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl. Acad. Sd. USA 88(17), 7595-7599.
  • oligonucleotides resistant to nuclease attack are for them to be "self-stabilised” as described by Tang et al (1993) Nucl. Acids Res. 21, 2729- 2735.
  • Self-stabilised oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and foetal bovine serum.
  • the self-stabilised region of the oligonucleotide does not interfere in hybridisation with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilised oligonucleotides with respect to their linear counterparts.
  • siKNA molecule and/or antisense oligonucleotide of the invention may comprise or consist of a sequence selected from those in the following table (which sequences are also described in the accompanying Examples):
  • the first agent is a compound with binding affinity for hCAP18/LL-37 and the second agent is a compound with binding affinity for EGF receptors such as proteins or carbohydrates.
  • the compound may bind substantially reversibly or substantially irreversibly to an active site of hCAP18/LL-37 and/or EGF receptors.
  • the compound may bind to a portion of hCAP18/LL-37 and/or EGF receptors that is not the active site so as to interfere with the binding of the hCAP18/LL-37 and/or EGF receptors to a ligand or receptor.
  • the compound may bind to a portion of hCAP18/LL-37 and/or EGF receptors so as to decrease the proteins activity by an allosteric effect.
  • This allosteric effect may be an allosteric effect that is involved in the natural regulation of the activity of hCAP18/LL-37 and/or EGF receptors, for example in the activation of the hCAP18/LL-37 and/or EGF receptors by an "upstream activator".
  • FRET Fluorescence Energy Resonance Transfer
  • a polypeptide that is labelled for example with a radioactive or fluorescent label.
  • a further method of identifying a compound that is capable of binding to the polypeptide is one where the polypeptide is exposed to the compound and any binding of the compound to the said polypeptide is detected and/or measured.
  • the binding constant for the binding of the compound to the polypeptide may be determined.
  • Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip-based method.
  • New technology, called VLSIPSTM has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location.
  • Bio chips have been produced in which each location has a scale of, for example, ten microns.
  • the chips can be used to determine whether target molecules interact with any of the probes on the chip.
  • scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.
  • yeast two-hybrid system Another method of identifying compounds with binding affinity for hCAP18/LL- 37 is the yeast two-hybrid system, where the polypeptides of the invention can be used to "capture" proteins that bind hCAP18/LL-37.
  • the yeast two-hybrid system is described in Fields & Song, Nature 340:245-246 (1989).
  • the compound has ligand-binding capacity for hCAP18/LL-37 and/or EGF receptors.
  • the first agent may be a soluble fragment of an hCAP18/LL-37 receptor (such as, but not limited to, FPRLl).
  • the agent may be a high affinity molecule that mimics an antibody (a so-called 'affibody') (for example, see US 5,831,012 and www.affibody.se).
  • 'affibody' a so-called 'affibody'
  • These ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A (a surface protein from the bacterium Staphylococcus aureus). This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein.
  • the first and/or second agent is a small inhibitor compound.
  • the first and/or second agent comprises or consists of a polypeptide.
  • the polypeptide is an antibody or an antigen-binding fragment thereof, preferably the antibody or antigen-binding fragment thereof is selected from the group consisting of Fv fragments, Fab-lilce fragments, single variable domains and domain antibodies.
  • the antibody or an antigen-binding fragment thereof is humanised.
  • antibody we include substantially intact antibody molecules, as well as chimaeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bispecif ⁇ c antibodies (for example, with affinity for both hCAP18/LL37 and an EGF receptor), antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen binding fragments and derivatives of the same.
  • antigen-binding fragment we mean a functional fragment of an antibody that is capable of binding to hCAP18/LL-37 and/or EGF receptors.
  • the antigen-binding fragment is selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments), single variable domains (e.g. V H and V L domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
  • Fv fragments e.g. single chain Fv and disulphide-bonded Fv
  • Fab-like fragments e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments
  • single variable domains e.g. V H and V L domains
  • dAbs including single and dual formats [i.e. dAb-linker-dAb]
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • modified versions of antibodies and an antigen-binding fragments thereof e.g. modified by the covalent attachment of polyethylene glycol or other suitable polymer.
  • Methods of generating antibodies and antibody fragments are well known in the art.
  • antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Set U.S.A. 86:3833-3837; Winter et al, 1991, Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture.
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982).
  • Antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York).
  • antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells ⁇ e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • antibody fragments can. be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • humanised antibodies are preferably used.
  • Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimaeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies.
  • Humanised antibodies include antibodies in which complementary deteraiining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat of rabbit having the desired functionality.
  • donor antibody such as mouse, rat of rabbit having the desired functionality.
  • Fv framework residues of the human antibody are replaced by corresponding non-human residues.
  • Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences.
  • the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determrning regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321:522-525; Eiechmann et al, 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).
  • the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain.
  • Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Nature 321:522-525; Reichrnann et al, 1988. Nature 332:323-327; Verhoeyen et al, 1988, Science 239:1534- 15361; US 4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity deteimining regions.
  • humanised antibodies are chrmaeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991, J. MoI. Biol. 227:381; Marks et al, 1991, J. MoI. Biol 222:581; Cole et al, 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al, 1991. J. Immunol. 147:86-95).
  • suitable antibodies may be tested for activity, for example by ELISA.
  • At least one of the first and second agents is capable of being selectively delivered to or selectively activated by the cancer cells.
  • hCAP18/LL-37 and/or EGF receptors By “selectively” we mean that the inhibitory action of the agent on the biological activity of hCAP18/LL-37 and/or EGF receptors is preferentially exerted at or within the cancer cells (other than by local administration of the agent to the site of cancer cells).
  • the first and/or second agent comprises a target cell specific portion, advantageously, the target cell specific portion is an antibody or antigen-binding fragment thereof which may be humanised.
  • target cell specific portion we mean the portion of the agent which comprises one or more binding sites which recognise and bind to entities on the target cancer cell. Upon contact with the target cell, the target cell specific portion may be internalised along with the inhibitor portion. The entities recognised by the target cell-specific portion are expressed predominantly, and preferably exclusively, on the target cancer cell. The target cell specific portion may contain one or more binding sites for different entities expressed on the same target cell type, or one or more binding sites for different entities expressed on two or more different target cell types.
  • the target cell-specific portion recognises the target cell with high avidity.
  • the antibody or antigen-binding fragment thereof has specificity for an antigen expressed on the surface of the cancer cell.
  • the entity which is recognised may be any suitable entity which is expressed by tumour cells. Often, the entity which is recognised will be an antigen.
  • antigens examples include those listed in Table 1.
  • Antigen 85A12 (Unipath) colon/rectum tumours.
  • HMFGl Polymorphic Epithelial HMFGl (Taylor- Imaging & Therapy of Mucin (Human milk fat Papadirnitriou, ICKF) ovarian cancer, pleural globule (Antis ' oma pic) effusions, breast, lung
  • CPG2 Gonadotropin
  • CD20 Antigen on B 1F5 (IgG2a) 3 Targeting of alkaline Lymphoma (normal and phosphatase. (Senter et neoplastic) al (1988) Proa Natl. Acad. Sd. USA 85, 4842-4846
  • antigens include alphafoetoprotein, Ca-125, prostate specific antigen and members of the epidermal growth factor receptor family, namely erb Bl (EGFR), erb B2, erb B3 and erb B4.
  • the second agent can be anti-EGF receptor antibody, such as an anti-erb B2 antibody ⁇ e.g. Herceptin), If fused or otherwise conjugated to the first agent, the second agent can act as both an EGF receptor inhibitor and a targeting agent.
  • an anti-erb B2 antibody e.g. Herceptin
  • the target cell-specific portion comprises two or more binding sites for the target cell, wherein the target cell specific portion is an antibody, or bivalent fragment thereof.
  • Said target cell specific portion may have respective 'arms' that recognise the same entity as one another or that recognise different entities.
  • the target cell specific portion has two 'arms' which recognise different molecules on the same target cell wherein the molecules on the same target cell are not confined to that cell type but may occur on a few other cell types.
  • one 'arm' of the target cell-specific portion may recognise molecules on cell types I, II and III, whereas the other 'arm' may recognise molecules on cell types I 5 IV and V.
  • an agent of the invention comprising such a target cell-specific portion will have greater specificity for cell type I compared with cell types II, III and IV.
  • This aspect of the invention is particularly helpful, as there have been very few completely target cell-specific molecules discovered, whereas molecules which occur on a few cell types, and which are useful in this aspect of the invention, are well known.
  • Such molecules are usually cell-surface antigens for which cross- reactive antibodies are known.
  • Monoclonal antibodies which will bind to many of the antigens listed in Table 1 are already known, but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens (see above).
  • the entity that is recognised may or may not be antigenic but can be recognised and selectively bound to in some other way.
  • it may be a characteristic cell surface receptor such as the receptor for melanocyte-stimulating hormone (MSH) which is expressed in high number in melanoma cells.
  • MSH melanocyte-stimulating hormone
  • the entity may be an entity that is induced in the target cells.
  • the cell-specific portion may then be a compound or part thereof which specifically binds to the entity in a non-immune sense, for example as a substrate or analogue thereof for a cell-surface enzyme or as a messenger.
  • the high avidity target cell specific portion comprises two or more different binding sites for the target cell.
  • the different binding sites for the target cell may or may not be two or more different antibodies, or fragments thereof, which are directed to different entities expressed on the target cell.
  • the different binding sites for the target cell may recognise and selectively bind the cell in some other, non-immune sense.
  • the targeting portion may be joined to the inhibitor agent of the invention by any suitable means which retains the functional activity of two portions.
  • the targeting portion and the inhibitor portion are both polypeptides, they may be fused to each other to create a fusion polypeptide. Examples of such fusions are well known to those skilled in the art.
  • the first and/or second agent is a prodrug selectively activated by the cancer cell.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to cancer cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form (see, for example, D.E.V. Wilman "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions 14, 375- 382 (615th Meeting, Harbor 1986) and VJ. Stella et al "Prodrugs: A Chemical Approach to Targeted Drug Delivery” Directed Drug Delivery R. Borchardt et al (ed.) pages 247-267 (Humana Press 1985)).
  • the adaptability of the strategy caters for the employment of a variety of enzymes which have the potential to release a multitude of mechanistically separate anticancer agents.
  • a single Mab-enzyme conjugate can generate therapeutically effective doses of mechanistically distinct anticancer agents possessing synergistic activities. This should prove important for immunogenicity reasons.
  • Enzymes of both mammalian and non-mammalian origin have been used for the activation of a wide range of prodrugs (Senter et al, 1993. Generation of cytotoxic agents by targeted enzymes. Bioconjugate 4, 3-9; Senter et al, 1991. Activation of prodrugs by antibody-enzyme conjugates. In Lnmunobiology of Proteins and Peptides VI, ed. M.ZAtassi. Plenum Press, New York, pp 97-105). While enzymes of mammalian origin might be advantageous due to reduced immunogenicity, the prodrugs that they act upon might be substrates for corresponding endogenous enzymes.
  • components (a) and (b) are suitable for sequential, separate and/or simultaneous use in the treatment of cancer cells.
  • the agents of the invention may be used to inhibit the proliferation of different types of cancer cell.
  • the cancer cells are epithelial cells and/or squamous cells
  • the cancer cells are selected from the group consisting of cancer cells of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands, gastrointestinal tract and endocrine system.
  • the cancer cells are breast cancer cells.
  • the cancer cells are estrogen positive.
  • the agents and methods of the invention may also be used to treat other cancer types.
  • the breast cancer cells are Elston grade III cells and may be metastatic.
  • a method of making a combination product as defined in the first aspect of the invention comprises bringing a component (a), as defined in the first aspect of the invention, into association with a component (b), as defined in the first aspect of the invention, thus rendering the two components suitable for administration in conjunction with each other.
  • kit of parts comprising:
  • the term "in conjunction with” includes that one or other of the two formulations may be administered (optionally repeatedly) prior to, after, and/or at the same time as, administration with the other component.
  • the terms “administered simultaneously” and “administered at the same time as” include that individual doses of the first agent that inhibits activity of hCAP18/LL-37 and the second agent that inhibits the activity of EGF receptors are administered within 48 hours (e.g. 24 hours) of each other.
  • a method of treatment of cancer cells which comprises administration of a combination product as defined in the first aspect of the invention or a kit of parts as defined in the third aspect of the invention to a patient suffering from, or susceptible to, cancer.
  • 'treatment' we include both therapeutic and prophylactic treatment of the patient.
  • 'prophylactic' is used to encompass the use of a polypeptide or formulation described herein which either prevents or reduces the likelihood of cancer in a patient or subject.
  • the method of treatment comprises the inhibition of proliferation and/or inhibition of metastasis of cancer cells in a patient.
  • the patient is human and advantageously the agent is selectively delivered to or selectively activated by the cancer cells.
  • the combination product is for use in the treatment of cancer.
  • a pharmaceutical composition comprising a combination product according to the first aspect of the invention and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is suitable for parenteral administration.
  • the formulation of the pharmaceutical composition is capable of targeted delivery of the agents to the cancer cells.
  • a combination product according to the first embodiment of the invention in the preparation of a medicament for inhibiting the proliferation of cancer cells.
  • the cancer cells are epithelial cells and/or squamous cells.
  • the cancer cells are selected from the group consisting of cancer cells of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.
  • the cancer cells are breast cancer cells, for example, Elston grade III cells and he cells may be metastatic.
  • 'pharmaceutical formulation means a therapeutically effective formulation according to the invention.
  • a 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity.
  • Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
  • a therapeutically effective amount of the active component is provided.
  • a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
  • agent or formulation thereof may be delivered as a single bolus dose (i.e. acute administration) or, more preferably, as a series of doses over time (i.e. chronic administration).
  • the agents of the invention can be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used and the indication for which it is being used.
  • the formulation comprises the agent of the invention at a concentration of between 0.1 ⁇ M and 1 mM, more preferably between 1 ⁇ M and 100 ⁇ M, between 5 ⁇ M and 50 ⁇ M, between 10 ⁇ M and 50 ⁇ M, between 20 ⁇ M and 40 ⁇ M and most preferably about 30 ⁇ M.
  • formulations may comprise a lower concentration of a compound of the invention, for example between 0.0025 ⁇ M and 1 ⁇ M.
  • agents of the invention will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19 th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).
  • the agents of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the agents of invention may also be administered via intracavemosal injection.
  • Such tablets may contain excipients such as macrocrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the agents of the invention can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, rntra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and 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 (lyophihsed) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously • described.
  • the daily dosage level of the agents of the invention will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.
  • the agents of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoro- methane, dichlorotetrafluoro-ethane, a hydro fiuoroalkane such as 1,1,1,2- tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoro- methane, dichlorotetrafluoro-ethane, a hydro fiuoroalkane such as 1,1,1,2- tetrafluoroethane (
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
  • Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the agents of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder.
  • the compounds of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route.
  • the agents of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylallconium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
  • the agents of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • the agent is a polypeptide
  • a sustained-release drug delivery system such as a microspheres. These are designed specifically to reduce the frequency of injections.
  • a sustained-release drug delivery system such as a microspheres.
  • rhGH recombinant human growth hormone
  • polypeptide agents of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site.
  • Electroporation therapy (EPT) systems can also be employed for the administration of proteins and polypeptides.
  • EPT Electroporation therapy
  • a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
  • Proteins and polypeptides can also be delivered by electroincorporation (EI).
  • EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
  • ReGeI thermo-sensitive ReGeI injectable. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
  • Protein and polypeptide pharmaceuticals can also be delivered orally.
  • One such system employs a natural process for oral uptake of vitamin B 12 in the body to co-deliver proteins and polypeptides. By riding the vitamin B 12 uptake system, the protein or polypeptide can move through the intestinal wall.
  • Complexes are produced between vitamin B 12 analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B 12 portion of the complex and significant bioactivity of the drug portion of the complex.
  • IF intrinsic factor
  • the constructs of the invention may be introduced into cells by methods involving retroviruses, so that the construct is inserted into the genome of the cell.
  • retroviral DNA constructs comprising a polynucleotide as described above may be made using methods well known in the art.
  • To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo* gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 ⁇ m pore- size filter and stored at -70°C.
  • the retrovirus For the introduction of the retrovirus into the tumour cells, it is convenient to inject directly retroviral supernatant to which 10 ⁇ g/ml Polybrene has been added. For tumours exceeding 10 mm in diameter it is appropriate to inject between 0.1 ml and 1 ml of retroviral supernatant; preferably 0.5 ml.
  • retrovirus-producing cells which produce retroviruses are injected.
  • the retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occurred within the tumour mass in situ.
  • proliferating cells can be successfully transduced in vivo if mixed with retroviral vector-producing cells.
  • Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre- existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).
  • MPB-PE N-[4-(p- maleimidophenyl)butyryl]-phosphatidylethanolamine
  • MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface.
  • the liposome is conveniently loaded with the agent of the invention (such as DNA or other genetic construct) for delivery to the target cells, for example, by forming the said liposomes in a solution of the agent, followed by sequential extrusion through polycarbonate membrane filters with 0.6 ⁇ m and 0.2 ⁇ m pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrif ⁇ gation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour.
  • the agent of the invention such as DNA or other genetic construct
  • adenoviruses carrying external DNA via an antibody-polylysine bridge see Curiel Prog. Med. Virol. 40, 1-18
  • transferrin-polycation conjugates as carriers
  • a polycation- antibody complex is formed with, an oligonucleotide agent of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody.
  • the polycation moiety binds the oligonucleotide agent via electrostatic interactions with the phosphate backbone.
  • the adenovirus because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the oligonucleotide agent of the invention. It is preferred if the polycation is polylysine.
  • the oligonucleotide agent may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.
  • a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations mat bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell.
  • the transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs).
  • complexes of transferrin-polycation and the DNA constructs or other genetic constructs of the invention are supplied to the tumour cells, a high level of expression from- the construct in the cells is expected.
  • High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Gotten et al (1992) Proc. Natl. Acad. ScL USA 89, 6094-6098 may also be used.
  • This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.
  • This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.
  • naked DNA and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the individual to be treated.
  • Non-viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144.
  • Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adeno virus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell- selective moiety into a fibre protein.
  • Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells such as those described in Bischoff et al (1996) Science 274, 373-376 are also useful for delivering the genetic construct of the invention to a cell.
  • a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention.
  • Other suitable viruses or virus-like particles include HSV, AAV, vaccinia and parvovirus.
  • the agent which selectively prevents the function of hCAJP18/LL-37 is a ribozyme capable of cleaving targeted hCAP18/LL-37 mRNA or DNA.
  • a gene expressing said ribozyme may be administered in substantially the same and using substantially the same vehicles as for antisense molecules.
  • Ribozymes which may be encoded in the genomes of the viruses or virus-like particles herein disclosed are described in US 5,180,818, US 5,168,053, US 5,149,796, US 5,116,742,US 5,093,246 and US 4,987,071. It will be appreciated that it may be desirable that the antisense molecule or ribozyme is expressed from a cell-specific promoter element.
  • the formulation is capable of targeted delivery of the agents of the invention to cancer cells.
  • agents and pharmaceutical formulations of the present invention have utility in both the medical and veterinary fields.
  • the agents of the invention may be used in the treatment of both human and non-human animals (such as horses, dogs and cats).
  • the patient is human
  • Figure 1 - hCAP18/LL-37 is highly expressed in breast cancer.
  • Figure 2 - h CAP-18/LL-37 is detected by immunoblotting in breast cancer.
  • Example 1-10 Clinical data of patients are presented in Table 2 (sample 1-10). Recombinant cathelin (C) and LL-37 peptide (L) were used as size references. Normal breast tissue is presented in lane 1. Elston grade I tumours are presented in lanes 2, 4 and 5. A grade II tumour is presented in lane 3 and grade III tumours are presented in lanes 6-10. In all tissues there were immunoreactive bands corresponding to the intact non-processed 18 IcDa holoprotein. The processed LL- 37 peptide (4 IcD) was visible in 4 of the 5 grade III tumours (no 7-10).
  • Figure 3 Transgenic expression of hCAPlS in epithelial cells increases cell proliferation.
  • Figure 4 Treatment with synthetic LL-37 peptide increases cell proliferation of epithelial cells.
  • HaCaT cells synchronized by serum starvation for 72 hours and then treated for 36 hours with 10 ⁇ g/ml of synthetic, biologically active ⁇ L-31 peptide (in DMEM + 5% FCS + PEST) show significantly increased cell proliferation compared with non treated (control) HaCaT cells, proliferation rate evaluated with [ 3 H]-Thymidine incorporation.
  • Figure 5 The LL-37 receptor FPRLl is expressed in breast cancer and in normal mammary gland epithelium.
  • Figure 6 Increased expression of hCAP18/LL-37 (displayed in logarithmic scale) in estrogen receptor (ER) and lymph node (N) positive breast tumours.
  • Figure 7 - LL-37 inhibits camptothecin-induced apoptosis in HEK Cells (A) and HaCat cells (B)
  • FIG. 8 Flow cytometry analysis of HEKn cells showing that pretreatment of HEKn cells with LL-37 protects from camptothecin-induced apoptosis
  • the cells were cultured in the presence or absence LL-37 and camptothecin and then harvested and analysed for caspase-3 activity by in vitro hydrolysis of VDVAD-AMC. Caspase activation induced by incubation for 24 hours with camptothecin was reduced by treatment the cells with LL-37. Values are means of three different experiments, each performed by triplicate.
  • HEKn cells were treated with 2 ⁇ M LL-37 and harvested at different time points after stimulation. RNA from these cells was reverse transcribed and the expression of IAP-2 was determined by real-time PCR. The transcription level of IAP-2 for each time point is shown normalized against 18S RNA and relative to the untreated cells (control set as 1 for each time point).
  • Figure 11 - LL-37 increases the Prostaglandin endoperoxide synthase-2 (COX-2) expression in HEK cells
  • HEKn cells were treated with 2 ⁇ M LL-37 and harvested at 6, ,12 and 24 hours after stimulation. Untreated cells were used as a control in each time point (light grey columns). RNA from these cells was reverse transcribed and the relative expression of COX-2 with respect to the control was determined by qPCR. as above.
  • HEKn cells were treated with or without the specific COX-2 inhibitor SC-791 (25nM) and further incubated for 6 hours with or without LL-37 (2 ⁇ M).
  • Levels of LAP -2 mRNA gene were analyzed by RT-PCR and are displayed relative to the untreated control sample.
  • Figure 13 - LL-37 potentiates the activation of the EGFR signaling system through Heregulin.
  • Extracts of the breast cancer line ZR75-1 were analysed by Western blot analysis against phosphorylated proteins as indicated.
  • LL-37 activates MAPK through the EGFR family, and the activation can be fully blocked by the tyrosine kinase inhibitor PD153035.
  • the required concentration for complete inhibition is 2.5 ⁇ M, which is higher than required for EGFR but necessary to block the activity of ERBB2.
  • Figure 16 - LL-37 inhibits metastasis of breast cancer cells
  • the tumorigenic potential of LL-37 was investigated in a colony formation assay using the breast cancer line ZR75-1.
  • LL-37 on its own decreased the number of colonies, whereas the combination of LL-37 and Heregulin increased the number of colonies (see Figure A). More dramatic was the phenotypic change.
  • the compact structure of the colonies was disrupted and singular cell satellites appeared (see Figure B). Heregulin restored the density of the colony, however not prohibiting the escape of singular cells when LL-37 was present.
  • C Expression of LL-37 in transfected ascites cells harvested from A SCID mouse injected with breast cancer cells transfected with and overexpressing hCAP18. Active transcription of hC AP 18 in MCF7 ascites cells (top plate) was confirmed by co-expression of a marker, green fluorescent protein (bottom plate).
  • siRNA short interfering RNA
  • siRNA-CAMPl-4 All four siRNA-CAMPs (siRNA-CAMPl-4; described in Example H) were pooled as a cocktail to minimize non-specific effects and similarly were done for the negative siRNA-Controls. Instructions for transfection optimization are provided in the detailed Protocol accompanying the siPORT transfection agent. All experimental procedures were done according to the manufacturers' descriptions. The final concentrations in siRNA were 1OnM, 25nM or 100 nM. Cell extracts of the treated breast cancer cells were analysed in western blot for hCAP18/LL-37 protein as described in M&M, Example C, using antibody against hCAPl ⁇ at 1/2000 dilution.
  • ECL Enhanced chemolumrniscence
  • Figure 20 Human antimicrobial protein hCAP18/LL-37 induces a metastatic phenotype in breast cancer that can be reversed by hCAP18 specific siRNA
  • the BioCoat Matrigel Invasion Chamber kit (Becton Dickinson Bioscience, Bedford, MA, USA) were used all according to the manufacturer's instructions. Before the transfer of cells to Matrigel filters, cells were starved in DMEM (Gibco-BRL) without FCS for 24h. The day cells were used they were trypsmized with 0.25% Tryp-EDTA (Invitrogen, cat#25200-056) and stopped by adding 50 ⁇ l trypsin inhibitor and lOO ⁇ l DMEM. 200 ⁇ l of a 220.000 cells/ml cell suspension were seeded into the transwell insert chamber with a filter coated with Matrigel and placed in the lower chambers filled with 750 ⁇ l of DMEM containing 5% FCS.
  • RNA interference by siRNA demonstrated a clear ability to inhibit the metastatic potential of cancer cells by reduction of the number of invasive tumor cell.
  • Example A The antimicrobial protein I ⁇ CAP18/LL-37 is highly expressed in breast cancer and is a putative growth factor for epithelial cells
  • hCAP18/LL-37 the expression pattern of hCAP18/LL-37 in a series of breast carcinomas is investigated, demonstrating a marked upregulation of hCAP18 mRNA and protein in the tumour cells but not in the adjacent stroma.
  • the highest levels of hCAPlS protein were detected among tumours with the highest histologic grade, whereas hCAP18 levels in some low grade tumours equalled those detected in the normal breast tissue.
  • Frozen tumour tissue from 28 breast cancer patients was obtained from the Department of Pathology, Danderyd Hospital, Sweden (Table 2). The tumours were scored according to Elston and Ellis I-IH, following established guidelines 13 . Cyclin A was used as proliferation marker (Nova-Castra Laboratories, Newcastle upon Tyne, UK). Estrogen receptor status was assessed on routinely processed paraffin sections. Uninvolved mammary tissue from eight patients with breast cancer and from two healthy individuals undergoing reductive breast surgery served as controls. All samples were examined by the same pathologist (B.S.) and classified as normal (Table 2). Written, informed consent was given by all patients. The study was approved by the Regional Committee of Ethics.
  • Frozen tumour tissues 16-60 mg, were homogenised in lysine buffer using an electric homogeniser. Proteins from tumour tissues and cell lines were extracted in SDS-containing sample buffer according to standard protocols 15 . The protein concentration was determined by a spectroph ⁇ tometric assay and adjusted with SDS-containing sample buffer to equal protein concentration 16 . For the detection of hCAP18/LL-37 the extracts were separated on 16.5% Tris-Tricine Ready gels (Bio-Rad Laboratories, Hercules, CA). Recombinant cathelin ⁇ and synthetic LL- 37 peptide were used as size references. For the detection of ERK1/2 and FPRLl, protein was separated on 12% and 8 % Tris-Glyci ⁇ e gels respectively.
  • the filters were reversibly stained with a 3% Ponceau S solution (Sigma Aldrich, USA) in 3% TCA, before incubating with the primary antibody.
  • Affinity purified anti-cathelin antiserum ⁇ , affinity-purified anti-LL-37 antiserum 10 , anti-FPRLl antiserum (scl8191, Santa Cruz Biotechnology, CA) and monoclonal anti- ERK1/2 antibody (Cell Signaling Technology, Beverly MA) were all used at 1:1000 dilution.
  • a sandwich ELISA previously described ⁇ was used to quantify hCAP18 in protein extracts from normal mammary gland and tumour tissues.
  • RNA from four normal samples and four tumours was extracted with the Qiagen RNeasy kit (Operon Biotechnologies, Cologne, Germany) and reverse transcribed with a first strand synthesis kit (Amersham Biosciences, Norwalk, CT). RNA was quantified by Real-Time PCR on an ABI Prism 7700 (Applied Biosystems) using 10 ng of cDNA according to standard protocols. The samples were evaluated in triplicates.
  • LL-37 peptide was synthesised and purified by HPLC to a purity of 98% (Polypeptide Laboratories A/S, Hiller ⁇ d, Denmark). Biological activity of the peptide was confirmed in an antibacterial assay 1S .
  • DMEM Dulbecco's modified Eagle's medium, Gibco BRL, Life Technologies, Scotland
  • FCS fetal calf serum, Hy- Clone, Boule Nordic AB Huddinge, Sweden
  • PEST penicillin 50 LVl and streptomycin 50 mg/ml, Gibco BRL
  • HEK293 and HaCaT cells were transfected using Fugene (Roche Diagnostics, Indianapolis, IN) under standard conditions, and selected for two weeks with 400 ng/ml G418 (Invitrogen, Paisley, UK).
  • HEK293 cells transfected with hCAP18 were harvested at 70% confluence and seeded in 24-well plates. After 24 hours, medium was changed and cells were cultured in 2 ml of medium (Optimem, Gibco BRL, Life Technologies, Scotland) supplemented with 5% FCS and PEST. Cells were harvested at day 6 and counted by flow cytometry (Becton Dickinson, Bedford, MA). Cell viability was measured with Trypan Blue; under all conditions less than 5% of the cells were Trypan Blue positive. All conditions were performed in triplicates. HEK293 cells transfected with the vector expressing only EGFP served as control.
  • HaCaT cells transfected with hCAP18 were harvested at 70% confluence and seeded at 2000 cell per well in 96 well plates in DMEM with 10% FCS + PEST. Medium was changed 12 hours later to DMEM supplemented with 5% FCS + PEST. After 24 hours of culture, the cells were treated 12 hours with 1 ⁇ Ci/well of [ 3 H]-Thymidine, harvested and analysed as described above. HaCaT cells transfected with the vector only expressing EGFP served as control.
  • RNA from HaCaT cells was extracted with the RNeasy kit (Qiagen) and reverse transcribed with a first strand synthesis kit (Amer sham-Pharmacia). FPPvLl RNA was quantified by Real-Time PCR and normalized against 18S-RNA as described above. Sequences were 5'-TCTGCTGGCTACACTGTTCTGC-S ' [SEQ ID N0:2] and 5'-GACCCCGAGGACAAAGGTG-S ' [SEQ ID N0:3] for the primers, and 6-FAM-5'- CCCAAGCACCACCAATGGGAGGA-3'-BHQl [SEQ ID NO:4] for the fluorigenic probe.
  • HaCaT cells were treated with the G-protein-coupled receptor inhibitor pertussis toxin.
  • Cells were preincubated with pertussis toxin (Sigma-Aldrich, Switzerland) 24 h before the LL-37 treatment in a final toxin concentration of 20 ng/ml.
  • Medium was changed 48 hours after cell seeding and the HaCaT cells were treated with 100 ⁇ l of medium (DMEM + 5% FCS and PEST) containing synthetic biologically active LL-37 peptide at 5 or 10 ⁇ g per ml respectively.
  • DMEM + 5% FCS and PEST synthetic biologically active LL-37 peptide
  • HaCaT cells were seeded at 10% confluence and kept in DMEM with 0.2% FCS for 36 hours. For the next 48 hours, cells were cultured in DMEM with 1% or 5% FCS respectively, and in presence or absence of LL-37 at lO ⁇ g/ml, with daily changes of medium. EGF at 10 ng/ml served as positive control. The expression of phosphorylated ERK 1/2 was evaluated by Western blot analysis with a mouse monoclonal antibody (Cell Signaling Technology, Beverly MA).
  • hCAP18/LL-37 is expressed in breast cancer
  • IJCAPI 8/LL-37 increases proliferation of epithelial cells
  • HEK293 and HaCaT cells transfected with an hCAP18 (hCAP18/E) expression vector demonstrated significantly higher proliferation rate than control cells transfected with the vector expressing EGFP only (E) (Fig. 3 A and B).
  • hCAP18/E hCAP18/E expression vector
  • FIG. 3 A and B By immunoblotting of protein extracts from the transfected HEK293 and HaCaT cells, we confirmed that these hCAP18 vector-containing cells produced the holoprotein (Fig. 3 A and B) and a 4 IcD immunoreactive band corresponding to LL-37 was detected in the cell medium (data not shown).
  • HaCaT cells cultured at 5% fetal calf serum and treated with synthetic biologically active LL-37 peptide at lO ⁇ g/ml demonstrated a significant increase in cell proliferation (Fig. 4).
  • TheLL-37 receptor FPRLl is expressed in breast cancer
  • FPRLl The G-protein-coupled receptor, FPRLl has been shown to mediate LL-37 induced effects in eulcaryote cells 4> 5 and to assess its potential role in the present setting, we investigated the expression of FPRLl protein in mammary tissue and found strong immunoreactivity for FPRLl both in breast cancer cells and in normal glandular epithelium (Fig 5a,b). Immunoblotting confirmed that FPRLl was expressed in both tissues (Fig. 5c). In addition, transgenic expression of I1CAPI8 significantly increased the expression of FPRLl mRNA (Fig. 5d) in HaCaT cells which may further support the involvement of FPRLl in hCAP18/LL-37 signalling.
  • HaCat cells pretreatment of HaCat cells with pertussis toxin did not abolish but suppressed the proliferation of these cells by approximately 50% (not shown), indicating that FPRLl may not be uniquely involved in mediating hCAP18/LL-37 growth stimulatory effects in these cells.
  • ERK1/2 the possible involvement of ERK1/2 in activation of epithelial cell proliferation, we treated HaCaT cells with synthetic biologically active LL-37 but there was no significant activation of ERK1/2, which indicates that EGFR is not involved in mediating the LL-37 stimulatory effect on HaCaT cell proliferation.
  • hCAP18/LL-37 is constitutively produced in normal mammary gland epithelium. This is consistent with a role for LL-37 in antimicrobial barrier protection in human and agrees with earlier reports where low constitutive expression of LL-37 was found in normal quiescent epithelium, in contrast to the pronounced expression seen in association with injury and inflammation 7"10 .
  • Constitutive expression of antimicrobial peptides has previously been detected in various exocrine glands such as the human cathelicidin LL-37 in sweat glands, the cathelicidin CRAMP in murine salivary glands and beta-defensins in human salivary glands 2I ⁇ 23 .
  • hCAP18 human beta-defensin-2 (hBD-2) mRNA in mammary glands was reported by BaIs et al in 1998 and recently other groups have found constitutive KBD-I expression in mammary glandular tissue of non-lactating women as well as in breast tissue during lactation and in breast milk 24"26 .
  • the production of hCAP18 was most notably increased in the breast epithelium of high grade tumours compared with normal mammary epithelium or low grade tumours.
  • the hCAPIS expression was however neither universal nor uniform, i.e. not all cancer cells were positive for hCAP18, but distinctly positive cells were found adjacent to cells devoid of detectable hCAPl ⁇ mRNA and protein (Fig. Ic), and the degree of expression varied considerably among cells in all tumour types. This may reflect a complex yet strictly controlled regulation of hCAP18 as has been suggested for human alpha-defensins in renal cell carcinoma 27 .
  • LL-37 exerts chemotactic effects in vitro, inducing migration of human neutrophils, monocytes, subsets of T-cells and mast cells Ai 35> 36 .
  • This chemotactic activity is dependent on binding of LL-37 to FPRLl 5 a pertussis toxin- sensitive, membrane bound G-protein-coupled receptor 4 .
  • Additional suggested functions for hCAP18/LL-37 include a role in epithelial repair and angiogenesis by promoting re-epithelialization of skin wounds and neovascularization 5> 10 .
  • the marked hCAP18/LL-37 expression in breast cancer cells presented herein reflects a growth, advantage for these tumour cells.
  • synthetic biologically active LL-37 peptide significantly increased proliferation of HaCaT cells.
  • LL-37 stimulates proliferation of epithelial cells, partially through FPRLl since blocking the receptor with pertussis toxin decreased the exogenous LL-37 proliferation effect by approximately 50%, possibly indicating the involvement also of other receptors.
  • Pathology NCGfBS Pathology reporting in breast cancer screening, second edition,NHSBSP Publication 1995.
  • Results are shown in Figure 6. The mean expression of the unaffected samples was arbitrarily set to 1. Mean and deviation were evaluated by Anova statistics.
  • hCAP18 The expression of hCAP18 is significantly higher (by about 5 times) in ER positive tumors when lymph nodes have developed, than without lymph nodes.
  • a number of well-defined in vitro and in vivo assays can be used to demonstrate different aspects of the contribution of hCAP18/LL-37 to tumour development, namely the induction of well-known signal transduction pathways, the stimulation of cell proliferation and suppression of apoptosis, the stimulation of colony growth and anchorage independent growth, the stimulation of invasivity through a basement membrane, and finally, the enhanced tumour growth and metastasis formation in mice.
  • a suitable target cell line is MCF-7, a low malignant estrogen receptor positive cell line that does not express hCAP18.
  • MCF-7 a low malignant estrogen receptor positive cell line that does not express hCAP18.
  • the synthesized and active protein LL-37 can be considered as a target for inhibition by antibodies.
  • the production of this protein can be hampered, by inhibiting the transcription of the hCAP18 gene via blocking of the promoter, as well as a posttranscriptional downregulation of the transcript by RNA interference.
  • experiments may be performed both in vitro, to demonstrate mechanism and target of action, and in vivo on tumours in the mouse, to demonstrate an effect in the body.
  • Such in vitro experiments may be performed on the MCF-7 derivatives described above.
  • cells are grown in Dulbecco's Modified Eagles medium containing 10% fetal calf serum.
  • Medium for the transgenic lines contains 150 ⁇ g/ml Hygromycin and 400 ⁇ g/ml Geneticin to maintain the transgenes.
  • the antibiotics are removed 24h before use of the cells, and the fetal calf serum concentration is adjusted to the concentration necessary for each particular experiment.
  • LL-37 is added at a concentration of 2 ⁇ M.
  • Transgenic cell lines and cell lines are harvested at 70% confluence and seeded at 2000 per well in 96-well plates in DMEM with 10% FCS. 24h later, medium is changed to medium containing 5% FCS with or without addition of LL-37. After 24h of culture, the cells are treated 12h with 1 ⁇ Ci/well of [3H] -thymidine, 20Ci/mmol, and harvested (Harvester 96; Tomtec, Orage, CT) onto a glass fiber filter (Wallac, Turku, Finland). The incorporation of [3 H] -thymidine is determined using a liquid scintillation counter (Microbeta Plus, Wallac). To ascertain statistic significance of the experiment, 24 wells are used for each condition/cell line.
  • This assay is performed as a complement to the [3H]-thymidine incorporation assay, to determine the stimulation of cell proliferation by hCAP18/LL-37.
  • the MCF-7 derivatives are plated at a density of 50 cells/mm2 (approx. 50 000 cells/well in a 6 well plate) in triplicate for each time point. The total cell number is quantified every 2 days with a hematocytometer. Cell viability is assessed by using trypan blue. Inhibition ofApoptosis by hCAP18/LL-37
  • the cells are seeded at 25000 cells/well in 6 well plates medium with 5% FCS, and treated with the topoisomerase I inhibitor camptothecin (CAM) (Sigma Chemical Co., St. Luis, MO) at 6 ⁇ M for 24h.
  • CAM topoisomerase I inhibitor camptothecin
  • the Propidium Iodide (PI)/RNase staining buffer (Becton Dickinson, San Jose, CA) is used for this analysis. Trypsinized cells are fixed in cold 70% (VfV) ethanol, and stored until its use at 4 0 C. The DNA content is measured through incorporation of PI into DNA. Fluorometric analysis is performed using a FACScan flow cytometer (Becton and Dickinson, Mountain View, CA, USA). FSC (forward light scatter) and SSC (side light scatter) of particles are simultaneously measured to determine the size and the granularity of cells. The red fluorescence of PI stained nuclei is detected in the FL-4 window (600 nm band pass filter and 35 nm band width).
  • the intensity of fluorescence is proportional to the cellular DNA content.
  • Each histogram is divided into two parts: (1) the Ml area representing asynchronous, non-apoptotic, live cells (cells in Gl 5 S and G2 phases with DNA content equal to 2N to 4N); and (2) the M2 area representing apoptotic cells with a smaller quantity of DNA in comparison to living cells.
  • Caspase-3 enzymatic activity is measured using the fluorometric substrate VDVAD-AMC (100 ⁇ M) and DEVD-AMC (50 ⁇ M).
  • Cell lysates are combined in a reaction buffer (100 mM HEPES, 10% sucrose, 5 mM dithiothreitol (DTT), 10- 6% NP-40, and 0.1% CHAPS, (pH 7.25) and added to a microliter plate.
  • the cleavage of the fluorogenic substrate is monitored by AMC liberation in a Fluoroscan II plate reader (Labsystems, Sweden). Fluorescence units are converted to pmol of AMC using a standard curve generated from free AMC. Data are analyzed by linear regression.
  • This assay is used to determine the capacity of a cell for sustained proliferation in absence of supporting, neighbouring cells. This property is considered to reflect the capacity of the cell to form tumours.
  • the transgenic cells are maintained under subconfluent conditions and trypsinized at 70% confluency at max, and are men diluted to a concentration of 5, 10, and 25 cells/ml growth medium. Cells are grown for 3 to 7 days (principally sufficient for 3 to 4 doublings), and the number of cells per colony is determined. If the recombinant plasmid expresses green fluorescent protein, cells and colonies can be counted in a fluorescence microscope. The result of colony formation is expressed as a distribution of fluorescent cells per colony, and a Wilcoxon's rank-sum test is used to compare distributions between the different transgenic lines, in presence and absence of LL-37 in the medium. Since MCF-7 cells are known to form rumours in presence of ⁇ -estrogen only, the effect of 1-5 nM estrogen to the growth of the transgenic cells can be assayed.
  • This assay reflects the capacity of the cell to form metastases.
  • Cells are prepared by treatment with a mixture of 300u/ml trypsin, 20u/ml elastase, and ImM EDTA. The presence of elastase facilitates the dissociation of cells into single cell suspensions.
  • Cells are suspended in growth medium and mixed 1:1 with growth medium containing molten 0.7% Seaplaque low melting temperature agarose, at a final concentration of 500 cells/ml and 0.35% agarose.
  • ImI of this mix are plated in 6 well plates, over a 2 ml layer of solidified medium/0.6% agarose. The cells are fed every 3-4 days be adding lOO ⁇ l of growth medium. After 2 weeks the top layer of the culture is stained with 0.2% p-iodonitrotetrazoliium violet (Sigma), and colonies larger than 100 ⁇ M in diameter are counted.
  • the assay is performed in triplicates, and repeated twice.
  • Matrigel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans) but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumour cells into Matrigel has been used to characterize the , involvement of extracellular matrix receptors and matrix degrading enzymes which play roles in tumour progression.
  • Matrigel is diluted to 2mg/ml in serum free cold growth medium, and 100 ⁇ l are placed into the upper chamber of a 24-well transwell and incubated at 37 0 C over night for gelling.
  • Cells are harvested and resuspended in medium containing 1% FCS at a density of 106/r ⁇ l. After washing the matrigel with warm serum free medium, 100 ⁇ l of the cell suspension is placed on top.
  • the lower chamber is filled with 600 ⁇ l of growth medium containing conditioned medium as chemoattractant. The chambers are incubated in the cell incubator for 6h to24h.
  • the transwells are stained (Diff-Quick staining solution, Fisher Scientific) and, after scraping off the noninvaded cells with a cotton swab, the invaded cells are counted in a light microscope.
  • Subconfluently grown cells are harvested and suspended in cold PBS at 2.5X 107 cells/ml. 200 ⁇ l portions are injected subcutaneously, into the fat pad, or into the tail vein of 4-6 week old female SCID mice. Since the tumour formation of MCF- 7 cells generally depends on estrogen, pellets have been implanted subcutaneous to release 1.7 mg ⁇ -estradiol per day over 60 days. The mice are palpated twice per week and the date when the first palpable tumour arises is recorded. Tumour growth is monitored manually, and mice are sacrificed latest at a tumour diameter of lcm. The number, size and distribution of tumours is recorded.
  • mice Small metastases are detected by sectioning the mice, and scanned to induce fluorescence from the GFP expressed from the recombinant plasmids.
  • spleen and liver are collagenized, and single cell suspensions are evaluated in a FACS sorter to determine the number of fluorescent cells.
  • vitamin D The strongest inducer of hCAPl ⁇ transcription presently known is vitamin D.
  • the vitamin D receptor directly activates hCAP18 transcription by binding to a response element in the hCAP18 promoter. Since thus small molecules can control the expression of hCAPIS, it is meaningful to systematically screen for inhibitory compounds.
  • Initial studies have shown that estrogen and some of its metabolites have no effect.
  • Vitamin A inhibits the transcription in skin keratinocytes but stimulates transcription in the breast cancer cell line ZR-75-1.
  • vitamin D such as ZK159222 (Schering AG), and TEI- 9647 (Tejin Institute for Medical Research, Tokyo), and antagonists to vitamin A such as AGN193109 (Allergen Pharmaceuticals), are potential agents for use in the methods of the invention.
  • ZR75-1 cells are plated at 25% confluency and treated with the potential inhibitor, dissolved in isopropanol or DMSO at 100 ⁇ M, at a final concentration of 100 nM.
  • RNA is extracted with the Qiagen RNeasy ldt (Operon Biotechnologies, Cologne, Germany) and reverse transcribed with a first strand synthesis kit (Amersham Biosciences, Norwalk, CT).
  • RNA is quantified by Real- Time PCR on an ABI Prism 7700 (Applied Biosystems) using 5 ng of cDNA according to standard protocols. The samples are evaluated in triplicates.
  • Sequences are 5'-GTCACCAGAGGATTGTGACTTCAA-S ' [SEQ ID NO: 2] and 5'- TTGAGGGTCACTGTCCCCATA-3' [SEQ ID NO: 3] for the primers, and 6-FAM-5'-CCGCTTCACCAGCCCGTCCTT-3'-BHQl [SEQ ID NO: 4] for the fluorigenic probe.
  • the samples are normalized by quantification of 18S- RNA (Assay on Demand, Applied Biosystems).
  • the activity of the promoter is determined by use of a recombinant plasmid in which the hCAP18 promoter controls a luciferase reporter gene.
  • ZR75-1 cells are plated at 25% confluency in 6-well plates, and transfected per well with 3 ⁇ g the reporter plasmid complexed with 6 ⁇ l of jetPEI (qBiogene). 14 h past transfection, the inhibitor is added as above. 24 h later, the cells are lysed and luciferase activity is measured in assay systems (Promega). The activities are normalized against ⁇ - galactosidase, expressed from 200ng of cotransfected plasmid (pEFl/lacZ, Invitrogen). Each experiment is performed at least twice and in triplicates in each assay.
  • RNA interference Today's most effective posttranscriptional inhibition is RNA interference, which basically induces a sequence specific and catalytic degradation of the target mJRNA.
  • the most effective target sites in a transcript can most easily be screened in a cell line into which short interfering RNA is transfected. The efficiency of RNA interference is monitored by quantitative PCR and Western blot analysis. A successful target site can then be expressed in a recombinant vector, designed to express the target molecule at the site of the tumour.
  • RNA A short interfering RNA is designed and synthesized on basis of the coding sequence of hCAPlS.
  • This RNA consists of a double-stranded 19-mer, plus a 2- base dTdT-overhang on either side.
  • siRNA databases can most simply be used in which target sites have been preselected (for hCAP18 e.g. siRNAs no. 14586, 14402, 146365 from Ambion).
  • ZR75-1 cells or transgenic MCF-7 cells are transfected with siRNA at a final concentration of 1OnM at max. to avoid an unspecific response, such as interferon-related pathways.
  • Control siRNA contains 2-3 mismatches in the target sequence.
  • hCAP18 is determined by RealTime PCR as described above, and by quantitative Western blot analysis.
  • protein is extracted in SDS-containing sample buffer, separated on a 15% Tris-Glyci ⁇ e gel and electroblotted onto nitrocellulose filters as described above. Filters are reversibly stained with 3% Ponceau S before incubating with affinity purified anti-LL-37 antiserum at 1/1000 dilution. Signals from HRP- conjugated secondary IgG using enhanced chemoluminiscence are captured and evaluated as described above.
  • mice are injected with MCF-7 cells expressing transgenic hCAP 18.
  • Half of the mice receive daily subcutaneous injections of 30mg inhibitor, dissolved in 50 ⁇ l olive oil (Sigma). The onset of tumour formation, size and distribution is assayed as above.
  • siRNA valid for in vivo approaches should downregulate hCAPl 8 expression by at least 90%.
  • hCAP18 expression For interfering with tumour formation in the mouse, the downregulation of hCAP18 expression needs to be maintained for several weeks. Daily injections of high amounts of siRNA into the tail vein have been proven possible but appear little realistic for therapeutic approaches.
  • the alternative is a stable expression of si RNA in the organism/rumour cell. Plasmids have been designed ⁇ e.g. pSuper, OriGene Inc) that express RNA as a hairpin, which then is i ⁇ tracellularly converted into siRNA.
  • the target siRNA sequence determined by above experiments is cloned into pSuper, and tumour cells are transfected with this construct prior to implantation into the mouse.
  • Therapeutic approaches i.e. the delivery of expression vectors past tumour formation, are presently not yet developed, but the delivery of plasmid with liposomes and the construction of retroviral and adenoviral expression vectors for this purpose is investigated worldwide.
  • RNAi An alternative to RNAi is the "classical" antisense approach.
  • Single-stranded oligonucleotides of 20-3bp in length, complementary to the target transcript are directly added into the cell medium or injected into the mouse.
  • the backbone of the oligonucleotide is usually modified, to inhibit its degradation and to facilitate its uptake without any carrier substrates.
  • the method requires a high dosage since the inhibitory mechanism is noncatalytic, and the backbone modifications increase the cytotoxicity and unspecific side effects.
  • a typical administration range is 100-500 ⁇ g/animal/day.
  • Chicken antibodies have been produced and affinity purified in large scale, sufficient for the planned experiments. Antibodies against LL-37 have previously been shown capable to inhibit the activity of LL-37. In vitro experiments are therefore not required.
  • tumours are induced in the mouse as above.
  • Antibodies are injected twice weeldy when tumours are palpable, using 1-lOOmg/kg antibody at the beginning.
  • Agarwal C et al: AGNl 93109 is a highly effective antagonist of retinoid action in human ectocervical epithelial cells. J Biol Chem 271: 12209-12212 (1996).
  • Rosier RN The proteosome inhibitor MGI32 attenuates retinoic acid receptor trans-activation and enhances trans-repression of nuclear factor B. Potential relevance to chemo-preventive interventions with retinoids. Molecular Cancer 3: 8-19 (2004).
  • Warburton C et al. Treatment of HER-2/neu overexpressing breast cancer xenograft models with trastuzumab (Herceptin) and gefitinib (ZD1839): drug combination effects on tumour growth, HER-2/neu and epidermal growth factor receptor expression, and viable hypoxic cell fraction. Clin Cancer Res. 10:2512-24 (2004).
  • Zhang M et al. Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer. Genet Vaccines Ther. 3:5-16 (2005).
  • Cathelicidins are a family of antimicrobial peptides found in many mammalian species. They consist of a highly conserved amino-terminal domain, cathelin, and a variable cafboxy-terminal domain which is released by proteolysis to confer the antimicrobial activity. (1,2).
  • the only human member of this family, 18kDa human cationic antimicrobial protein (hCAP18) is produced mainly by neutrophils, several epithelial and mucosal cells (skin, bronchi, buccal mucosa, sophagous, cervix, vagina, epidimus and salivary glands) (3-8).
  • the C-terrninal 37 amino acid domain, LL-37 displays antimicrobial activity against a broad spectrum of microorganisms (9,10) through disruption of the membrane stability (11,12).
  • this peptide has been implicated in other biological activities, such as chemotaxis and cytokine release (8,13) in blood and epithelial cells, induction of epithelial cell proliferation (14) and angiogenesis (15).
  • chemotaxis and cytokine release (8,13) in blood and epithelial cells
  • induction of epithelial cell proliferation (14)
  • angiogenesis 15
  • Recent studies have shown that LL-37 is highly expressed during wound healing, affects the in vitro proliferation of human keratinocytes and is involved in re-epithelization of wounds (16,17).
  • Foreskin Epidermal keratinocytes were obtained from Cascade Biologies (Cascade Biologies, Eugene, OR) and cultured in Epilife basal Medium (Cascade Biologies,. ) supplemented with 0,06 mM Calcium, 0.2%v/v BPE, 5 ⁇ g/ml bovine insuline, 0.18 ⁇ g/ml hydrocortisone, 5 ⁇ g/ml bovine transferrin, 0.2 ng/ml human epidermal growth factor, 100 U/ml penicillin G,100 ⁇ g/rnl streptomycin sulfate, and 0.25 ⁇ g/ml Ampotericin B (all Cascade Biologies) at 37°C and 5% CO2. Cells were passaged weekly using 0.025% (w/v) trypsin and 0.01% (w/v) EDTA (Cascade Biologies).
  • HaCaT keratinocytes were cultivated in DMEM (Dulbecco's modified Eagle's Medium; Gibco-BRL Technology, Paisley, UK) supplemented with 10% foetal calf serum (hyClone, Boule Nordic AB Huddinge, Sweden), 2 mM glutamine, penicillin (50 U/L Gibco-BRL) and streptomycin (50 mg/ml, Gibco-BRL).
  • DMEM Dulbecco's modified Eagle's Medium; Gibco-BRL Technology, Paisley, UK
  • foetal calf serum hyClone, Boule Nordic AB Huddinge, Sweden
  • penicillin 50 U/L Gibco-BRL
  • streptomycin 50 mg/ml, Gibco-BRL
  • 25,000 cells/well were plated in 6-well plate culture dishes. 48 after plating, cells were treated with 0, 0.23, 0.46, 0.69, 0.9, 1.15, 2.3 4.6 and 11.5 ⁇ M LL-37, the sequence of which is shown below.
  • the peptide was diluted in medium (DMEM 5% FCS to HaCaT cells and Epilife basal Medium supplemented with 0.1% FCS to HEKn cells). The cells were treated during a total time of 24h. After LL-37 stimulation, the topoisomerase I inhibitor camptothencin (CAM) (Sigma Chemical Co., St. Luis, MO) in dimethylsulfoxide was added to the cells to a final 6 ⁇ M concentration. Cells were further incubated for 24h before analysis.
  • DMEM 5% FCS to HaCaT cells and Epilife basal Medium supplemented with 0.1% FCS to HEKn cells
  • the PI/RNase staining buffer (BectonDickinson) was used for this analysis. Trypsinized cells were fixed in cold 70% (v/v) ethanol, and stored until its use at 4 °C. The DNA content was measured through incorporation of propidium iodide into DNA. Fluorometric analysis was performed using a FACScan flow cytometer (Becton and Dickinson, Mountain View, CA, USA). FSC (forward light scatter) and SSC (side light scatter) of particles were simultaneously measured to determine the size and the granularity of cells. The red fluorescence of PI stained nuclei was detected in the FL-4 window (600nm band pass filter and 35 nm bandwidth).
  • the intensity of fluorescence was proportional to the cellular DNA content.
  • Each histogram was divided into two parts: (1) the Ml area representing asynchronous, non-apoptotic, live cells (cells in Gl, S and G2 phases with DNA content equal to 2n to 4N); and (2) the M2 area representing apoptotic cells with a smaller quantity of DNA in comparison to living cells.
  • Caspase-3 enzymatic activity was estimated using the fluorometric substrate VDVAD-AMC (100 ⁇ M) and DEVD-AMC (50 ⁇ M) as previously described. Briefly, cell lysates were combined in a reaction buffer (100 mM HEPES, 10% sucrose, 5 mM dithiothreitol (DTT), 10-6% NP-40, and 0.1% CHAPS, (pH 7.25) and added to a microliter plate. The cleavage of the fluorogenic substrate was monitored by AMC liberation in a Fluoroscan II plate reader (Labsystems, Sweden). Fluorescence units were converted to pmol of AMC using a standard curve generated from free AMC. Data were analyzed by linear regression and are displayed as pmol AMC release per mm.
  • LL-37 inhibits the induction of Caspase-3 by CAM in HaCaT arid HEKn cells
  • caspase-3 is one of the key proteins in the apoptosis pathway and it is an early indicator of apoptosis
  • caspase-3 activity was significantly increased in keratinocytes within the first 12 hours of CAM treatment and peaked at 24 hours.
  • Pre-treatment with 2.3 ⁇ M of LL-37 decreased camptothencin-induced caspase activation (figure 9a and b).
  • LL-37 induces IAP-2 expression in human keratinocytes
  • IAP apoptosis proteins
  • the present data show that LL-37 is also involved in promoting cell survival in keratinocytes via an inhibitory effect on apoptosis.
  • hCAP18/LL-37 human cathelicidin antimicrobial peptide 18 kDa/LL-37; IAP-2, Inhibitor apoptosis protein-2; COX-2, ciclooxygenase-2; VDVAD-AMC, caspase 3 substrate; HEKn, human epidermal keratinocytes neonatal; DMEN, Dulbecco's Modified Eagle's Medium; PBS, phosphate buffered saline.
  • DEVDase Asp-Glu- Val-Asp protease activities
  • CHAPS 3-[(3-cholamidopropyl) dimethyl- ammmonio] propane- 1-sulphonic acid, CAM: Camptothecin
  • the low malignant breast cancer line ZR75-1 (obtained from ATCC) is used throughout the study.
  • ZR75-1 expresses both hCAPl ⁇ and ERBB2 as a low level.
  • LL-37 was synthesised in house.
  • Recombinant Heregulin- ⁇ (EGF domain, ac 178- 241) was obtained from Upstate, Lake Placid, NY.
  • Cells were washed with ice-cold PBS containing phosphatase inhibitors (1 mM NaF + 1 mM Na 3 VO 4 ), plus 2mM PMSF as protease inhibitor, and lysed with 200 ⁇ l SDS sample buffer containing the inhibitors as above.
  • the samples were homogenised and denatured at 80° C, 50 ⁇ l of sample was then loaded onto a 7.5% SDS-PAGE gel, separated and blotted onto nitrocellulose by standard procedures.
  • pERBB2 pTyr 1248, rabbit ,Upstate (cat no. 06-229), used 1/2000 - can be reused.
  • pMAPK 1/2 pThr 202/pTyr 204) , Cell Signalling Inc.
  • Chemoluminescence signals were normalised against Ponceau staining.
  • the tyrosine kinase inhibitor PD153035 (Merck Biosciences) was added at increasing concentrations from 20 nM to 2.5 ⁇ M.
  • Fig 13 displays the activation of ERBB2 and MAPK in ZR75-1 cells after treatment with LL-37, Hereguliu, or in combination.
  • Cell extracts were analysed by Western blot against phosphylated ERBB2 and MAPK. The signal was captured in a CCD camera and quantified after normalization against Ponceau staining. The evaluation of triplicates is shown in Fig. 14, indicating that LL-37 and Heregulin cooperate on the activation.
  • ERBB2 phosphorylation was found to be inhibited by the tyrosine kinase inhibitor PD153035, indicative of a role of MAPK in signalling.
  • PD153035 tyrosine kinase inhibitor
  • This assay reflects the capacity of the cell to form metastases.
  • Cells are prepared by treatment with a mixture of 300u/ml trypsin, 20u/ml elastase, and ImM EDTA. The presence of elastase facilitates the dissociation of cells into single cell suspensions.
  • Trypsinized cells are passed through a cell strainer and suspended in medium (containing double concentrated additions) at a final concentration of 1000 cells/ml.
  • the cell suspension is warmed to 37°C and mixed 1:1 with 0.7% agarose solution prepared as above. 2ml are put as a layer over the bottom agar and left 30min at RT to solidify the top layer.
  • the plates are placed into the incubator and cells are fed every 3-4 days by adding lOO ⁇ l of growth medium. After 2 weeks the top layer of the culture is stained with 0.2% p-iodonitrotetrazoliium violet (Sigma), and colonies larger than 100 ⁇ M in diameter are counted.
  • the assay is performed in triplicates, and repeated twice.
  • PenicillinG/streptomycrn (when working with ZR75-1 line): 1% stock solution
  • Results are shown in Figure 16.
  • the effect of LL-37 on tumorigenicity was investigated in vitro by assaying its impact to colony formation of breast cancer line ZR75-1 in soft agar. The effect was heavily modulated by the presence of other factors such as FCS and Heregulin.
  • Fig 16a shows the effect of the number of colonies at 2% FCS. In absence of Heregulin, LL-37 as such significantly decreased the number of colonies. The negative effect of LL-37 was entirely removed in presence of Heregulin, indicating that their functional cooperation is required.
  • LL-37 and Heregulin dramatically affected the appearance of the soft agar colonies.
  • Fig 16b shows a representative example of soft agar colonies, obtained after 14 days of cultivation and living cell staining. Exposure of LL-37 on its own resulted on a weaker staining of the colony and a less compact appearance, indicating a strong reduction of the colony density. Instead, a corona of singular cells appeared around the maternal clone. Heregulin on its own had little effect. When Heregulin and LL-37 were combined, the maternal colony was stained in a similar manner as the untreated colonies. However, the LL-37-induced corona of single cells was maintained.
  • LL-37 appears not to contribute to the growth of the primary tumour but to crucially stimulate its potential to produce metastases.
  • An inhibitor to LL-37 should therefore reduce the production of metastases in breast cancer, Metastases, and not the primary tumour, usually are the cause of death in breast cancer.
  • Example G Effect ofLL-37 on metastasis of breast cancer cells in vivo
  • the cell lines MJ 1117 [kindly provided from M Egeblad; see Int J Cancer 86, 617-625 (2000)] is a derivative of MCF7 that expresses ERBB2 from an episomal expression vector.
  • MJ 1005 is the corresponding control line equipped with the empty control vector.
  • the cell lines were cultivated in Optimem, 5-10% FCS 3 plus hygromycin B, 150 ⁇ g/ml.
  • Cells were sorted for EGFP expression with a MoFlo® high speed cell sorting flow cytometer (DakoCytomation, Fort Collins, CO) using SummitTM software for data analysis, and their expression of CAP 18 was quantified by immunoblotting. Control cell lines were similarly established by transfection with the vector expressing EGFP only. The cell lines maintained a stable expression of CAP 18 during several months of continued cultivation without any selection.
  • Cells were grown in Optimem/10%FCS/hygror ⁇ ycin 150 ⁇ g/ml/ G418 400 ⁇ g/ml. Antibiotics were removed 24h before harvest at 50% confluency. Cells were trypsinized and suspended in PBS/lmM MgC12 at 50 million/ml. 10 million cells (200 ⁇ l) were injected subcutaneously into the mouse. Since MCF7 cells require ⁇ -estrogen for tumour formation,.
  • Depository pills (1.7mg/day) had been implanted into the mice the day before infection. Mice were observed on a daily basis, and palpated for tumour formation twice per week. Palpable infiltration at site of injection was observed already 1 week after injection, but a continuously growing tumour was evident after appr 40 days. Mice were sacrificed when the tumour had reached approx. lcm 3 in size. AU tumours, either at the site of injection or spread in the mouse, were excised or snap frozen. In addition, spleen and liver were homogenized, and, after lysis of erythrocytes with 0.17% NH4C1 and removal of debris by centrifugation, analyzed for the presence of EGFP expressing cells by FACS analysis.
  • Fig 18a shows an example of a tumour developed in a SCID mouse from the control line MJ1005 IRES, a derivative of MCF7. No secondary tumor, or metastatic cells in liver of spleen, were hitherto detected in the control mice.
  • Fig 18b shows the effect of transgenic expression of hCAP18 in the same cell Line.
  • the primary tumor does not grow more aggressively growth than from the control line.
  • secondary tumours appear in multiple locations, among which lymph nodes neighbouring the primary tumor and in the opposite flank, and large abdominal masses.
  • Ascites fluid (fig 18c) contained high amounts of cells expressing green fluorescent protein, which is produced simultaneously as the hCAP18 transgene.
  • 3/4 of the SCID mice infected with the hCAP18 producing line have shown either metastatic tumours or the spread of metastatic cells to spleen and liver.
  • siRNA short interfering RNA
  • siRNA-CAMPs were pooled as a cocktail to minimize non-specific effects and similarly were done for the negative siRNA-Controls. Instructions for transfection optimization are provided in the detailed Protocol accompanying the siPORT transfection agent. All experimental procedures were done according to the manufacturers' descriptions. The final concentrations in siRNA were 1OnM, 25nM or 100 nM. Cell extracts of the treated breast cancer cells were analysed in western blot for hCAP18/LL-37 protein as described in M&M, Example C, using antibody against hCAPl ⁇ at 1/2000 dilution.
  • ECL Enhanced chernoluminiscence
  • the Matrigel Invasion Assay was performed to assess the metastatic potential of MCF7-hCAP18 transgene cells over-expressing the hCAP18/LL-37 protein compared to hCAP18/LL-37 non-expressing MCF7-IRES transgene control cells. (See M&M Transgenic expression of hC AP 18 in MCF7 derivatives in Example G).
  • the BioCoat Matrigel Invasion Chamber kit (Becton Dickinson Bioscience, Bedford, MA, USA) were used all according to the manufacturer's instructions. Before the transfer of cells to Matrigel filters, cells were starved in DMEM (Gibco-BRL) without FCS for 24h. The day cells were used they were trypsinized with 0.25% Tryp-EDTA (Invitrogen, cat#25200-056) and stopped by adding 50 ⁇ l trypsin inhibitor and lOO ⁇ l DMEM. 200 ⁇ l of a 220.000 cells/ml cell suspension were seeded into the transwell insert chamber with a filter coated with Matrigel and placed in the lower chambers filled with 750 ⁇ l of DMEM containing 5% FCS.
  • RNA interference by siRNA demonstrated a clear ability to inhibit the metastatic potential of cancer cells by reduction of the number of invasive tumor cell.
  • An anti-hCAP18/LL-37 antibody is expressed in NSO myeloma cells.
  • anti-erbB2 antibody is expressed in separate NSO myeloma cells.
  • NSO myeloma cells are co-transfected by electroporation with expression vectors encoding the constituent light and heavy chains of the fusion protein. Transfectomas are then selected and screened for antibody production by ELISA assays.
  • the antibodies are formulated into an aqueous sterile injection solution.
  • the formulation is then administered into patients suffering from breast cancer by intravenous (IV) infusion over 90 minutes.
  • IV intravenous
  • the dose is selected according to the individual requirements of each patient, as determined by the medical practitioner. Typically, however, an initial dose of 4mg/kg is used followed by weekly maintenance doses of 2 mg/kg.

Abstract

La présente invention concerne un produit combiné comprenant un premier agent (A) qui inhibe l'activité biologique de la protéine cationique antimicrobienne humaine hCAP18/LL-37 ; et un second agent (B) qui inhibe l'activité biologique d'un récepteur à l'EGF (facteur de croissance épidermique), chacun des composants (A) et (B) étant formulé en mélange avec un adjuvant, diluant ou véhicule acceptable du point de vue pharmaceutique. Dans un mode de réalisation préféré, le ou les agents altèrent la transcription, la traduction et/ou les propriétés de liaison de la protéine hCAP18/LL-37 et/ou d'un récepteur à l'EGF. De préférence, le ou les agents sont sélectionnés dans le groupe constitué de molécules d'ARN interférent court (ARNic), d'oligonucléotides antisens et de composés présentant une affinité de liaison pour la protéine hCAP18/LL-37 et/ou les récepteurs à l'EGF. L'invention concerne en outre des procédés servant à inhiber la prolifération et/ou la métastase de cellules cancéreuses chez un patient, ainsi que des compositions pharmaceutiques destinées à être utilisées dans le traitement d'un cancer.
EP07732700A 2006-05-04 2007-05-04 Produit de thérapie combinée et utilisations de celui-ci Withdrawn EP2012823A2 (fr)

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