WO2011158019A1 - Vaccin à base de polypeptides - Google Patents

Vaccin à base de polypeptides Download PDF

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Publication number
WO2011158019A1
WO2011158019A1 PCT/GB2011/051096 GB2011051096W WO2011158019A1 WO 2011158019 A1 WO2011158019 A1 WO 2011158019A1 GB 2011051096 W GB2011051096 W GB 2011051096W WO 2011158019 A1 WO2011158019 A1 WO 2011158019A1
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Prior art keywords
fragment
composition according
pappalysin
variant
peptide
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PCT/GB2011/051096
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English (en)
Inventor
Jennifer Carling-Wright
Richard Birnie
Andrew William Heath
Norman Maitland
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Adjuvantix Limited
Procure Therapeutics Limited
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Priority claimed from GBGB1010054.3A external-priority patent/GB201010054D0/en
Priority claimed from GBGB1010058.4A external-priority patent/GB201010058D0/en
Priority claimed from GBGB1010056.8A external-priority patent/GB201010056D0/en
Application filed by Adjuvantix Limited, Procure Therapeutics Limited filed Critical Adjuvantix Limited
Publication of WO2011158019A1 publication Critical patent/WO2011158019A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001158—Proteinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/884—Vaccine for a specifically defined cancer prostate

Definitions

  • vaccine compositions comprising fragments and peptides derived from a pappalysin polypeptide and the use of the vaccine in the prevention and treatment of cancer, for example prostate cancer.
  • Pappalysin is a secreted pregnancy associated metalloproteinase of molecular weight 181 kilodaltons which naturally exists as a disulphide linked homodimer which is expressed continually during pregnancy and is found in a complex with an inhibitor protein called eosinophil major basic protein in a 2:2 proteinase:inhibitor complex.
  • a second form of the enzyme exists as pappalysin 2 [PappA2] which has a molecular weight of 198.5 kilodaltons, functions as a monomer and is preferentially expressed in the placenta and non pregnant mammary gland with low expression in the kidney, fetal brain and pancreas.
  • the substrates for pappalysin are insulin like growth factor binding proteins [IGFBP] of which there are 6 different proteins.
  • IGFBP 4 and 5 are the preferred substrates for pappalysin.
  • PappA2 cleaves IGFBP 5 preferentially.
  • IGFBPs are found tightly bound with insulin-like growth factor [IGF-1 ] which inhibits IGF-1 activity.
  • IGF-1 is a 70 amino acid polypeptide with a molecular weight of 7.6kDa. IGF-1 stimulates, amongst other cells, the proliferation of chondrocytes resulting in bone growth. IGF-1 is also implicated in muscle development.
  • IGF-1 is an example of a protein ligand that interacts with members of the receptor tyrosine kinase (RTK) superfamily. Approximately 98% of IGF-1 is bound to one of the six IGFBPs. IGFBP3 is the most abundant and accounts for 80% of IGF-1 binding. IGF-1 binds two receptors; the IGF-1 receptor (IGFR) and insulin receptor (IR) the former of which is bound with greater affinity.
  • IGFR IGF-1 receptor
  • IR insulin receptor
  • WO2005/089043 describes the isolation of prostate stem cells which have been directly isolated from lymph node and prostate glands from a series of patient samples. These stem cells express markers that characterise the cells with stem cell properties. The following markers are typically expressed as prostate stem cell markers; human epithelial antigen (HEA), CD44, a 2 3i hl and CD133. Furthermore, array expression of genes that are up regulated in cancer prostate stem cells when compared to normal prostate stem cells shows that one of the most highly up regulated genes in the array is pappalysin.
  • An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells.
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid.
  • Such antigens contain B-cell epitopes but no T cell epitopes.
  • the receptor CD40 plays an important co-stimulatory role in the activation of B-cells during the cognate interaction of antigen-specific T and B-cells that gives rise to an antibody response.
  • the CD40 signal is pivotal to the expression of T cell help and immunoglobulin class-switching in both humans and mice.
  • ligation of CD40 is also very important in activation of macrophages and of dendritic cells to express co-stimulatory antigens and thus in the generation of helper T cell priming by these antigen-presenting cells.
  • This disclosure relates to the combination an antigen derived from a pappalysin polypeptide and a ligand that binds CD40 receptor, typically a CD40 adjuvant monoclonal antibody, alternative adjuvants to a CD40 monoclonal antibody and combinations of adjuvants that include CD40 monoclonal antibodies and their use in a cancer vaccine.
  • This disclosure also relates to the identification of CTL peptide epitopes within a pappalysin and vaccines comprising said CTL peptide[s].
  • a vaccine or immunogenic composition comprising:
  • pappalysin or a polypeptide fragment derived from a pappalysin
  • a vaccine or immunogenic composition wherein said composition comprises:
  • CD40 adjuvant monoclonal antibody or CD40 adjuvant antibody binding fragment thereof, linked to a pappalysin, or pappalysin polypeptide fragment;
  • pappalysin peptide fragment derived from pappalysin and an adjuvant wherein said adjuvant is not a CD40 monoclonal antibody, or CD40 antibody fragment.
  • a vaccine or immunogenic composition comprising an isolated peptide antigen wherein said peptide antigen is less than 50 amino acids in length and specifically binds an antibody that binds a pappalysin and including an adjuvant and/or carrier.
  • a vaccine or immunogenic composition comprising a polypeptide derived from a human pappalysin wherein said polypeptide is not full length mature human pappalysin and comprises a CTL peptide epitope wherein said peptide epitope is 8 or 9 amino acids in length and including an adjuvant and/or carrier.
  • said pappalysin is human pappalysin 1 .
  • said pappalysin is represented by the amino acid sequence in Figure 1 .
  • said fragment is a peptide.
  • said peptide is 10-30 amino acids in length; preferably said peptide is 8-18 amino acids in length.
  • said peptide is 10 amino acids in length.
  • said peptide is 29 amino acids in length.
  • said pappalysin fragment is represented by the amino acid sequence in Figure 2, or a variant fragment comprising a variant sequence wherein said variant sequence is modified by addition, deletion or substitution of one or more amino acid residues and wherein said variant fragment retains or has enhanced immunogenicity when compared to a non variant sequence.
  • said fragment comprises or consists of one or more peptides selected from the group consisting of:
  • said pappalysin fragment is represented by the amino acid sequence in Figure 3 or a variant fragment comprising a variant sequence wherein said variant sequence is modified by addition, deletion or substitution of one or more amino acid residues and wherein said variant fragment retains or has enhanced immunogenicity when compared to a non variant sequence.
  • said fragment comprises or consists of one or more peptides selected from the group consisting of: LYEDDHKNPTVTREQVD;
  • said pappalysin fragment is represented by the amino acid sequence in Figure 4 or a variant fragment comprising a variant sequence wherein said variant sequence is modified by addition, deletion or substitution of one or more amino acid residues and wherein said variant fragment retains or has enhanced immunogenicity when compared to a non variant sequence.
  • said pappalysin fragment is represented by the amino acid sequence in Figure 5 or a variant fragment comprising a variant sequence wherein said variant sequence is modified by addition, deletion or substitution of one or more amino acid residues and wherein said variant fragment retains or has enhanced immunogenicity when compared to a non variant sequence.
  • said pappalysin fragment is represented by the amino acid sequence in Figure 6 or a variant fragment comprising a variant sequence wherein said variant sequence is modified by addition, deletion or substitution of one or more amino acid residues and wherein said variant fragment retains or has enhanced immunogenicity when compared to a non variant sequence.
  • said pappalysin fragment is represented by the amino acid sequence in Figure 7 or a variant fragment comprising a variant sequence wherein said variant sequence is modified by addition, deletion or substitution of one or more amino acid residues and wherein said variant fragment retains or has enhanced immunogenicity when compared to a non variant sequence.
  • said fragment comprises or consists of one or more peptides selected from the group consisting of:
  • FMGDNYCDAINNRA wherein said fragment includes one or more pappalysin epitopes.
  • peptide is a variant peptide of i) modified by addition, deletion or substitution of at least one amino acid residue wherein said variant peptide retains or has enhanced immunogenicity when compared to a non-variant peptide.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: RLWSWVLHL; TEEPSPPSR; DLELPRDAF; YIEHFSLWK; SLEPPLCGQ; VNELKNILK; ETEPSFETG; ESEPSPAVT; CIDEPSRCY; ACEPVDCSI; TCDPPPPKF; or GCEPFMGDN.
  • a CTL peptide epitope selected from the group: RLWSWVLHL; TEEPSPPSR; DLELPRDAF; YIEHFSLWK; SLEPPLCGQ; VNELKNILK; ETEPSFETG; ESEPSPAVT; CIDEPSRCY; ACEPVDCSI; TCDPPPPKF; or GCEPFMGDN.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: RLWSWVLHL; ELPRDAFTL; KDPRYFFSL; KLYVNGAQV; KQYNISWEL; VLNPSFYGM; RMHCYLDLV; ILVQYASNA; RLWDVGEEV; QLAQTTFWL; PMVAAAVIV; LLTCMEDG; GLWSFPEAL; TWNGSFHV; LKWYPHPAL; or G TWNGSFHV.
  • a CTL peptide epitope selected from the group: RLWSWVLHL; ELPRDAFTL; KDPRYFFSL; KLYVNGAQV; KQYNISWEL; VLNPSFYGM; RMHCYLDLV; ILVQYASNA; RLWDVGEEV; QLAQTTFWL; PMVAAAVIV; LLTCMEDG; GLWSFPEAL; TWNGSFHV; LKWYPHPAL; or G TW
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: AVITGLYDK; ATYDGQFMK; GIFSPLTQK; QLSSFRQPK; ILANCDISK; HLRHPAFVK; GVATWPWDK; GVCEEFEQK; or GLYQCTNGF.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: EPSPPSRAL; SGRGEQLRL; VARTQREIL; EPPLCGQTLVVRYRVVNL; EPSFETGDL; CPEPQGCYL; EPQGCYLEL; AVSGKNISLVVRDPPLQM; GVYTPQGFL; VCRTKVIDL; QPMVAAAVI; VPNELNSNL TVRDIPHWL; or TPFPMSCDL.
  • a CTL peptide epitope selected from the group: EPSPPSRAL; SGRGEQLRL; VARTQREIL; EPPLCGQTLVVRYRVVNL; EPSFETGDL; CPEPQGCYL; EPQGCYLEL; AVSGKNISLVVRDPPLQM; GVYTPQGFL; VCRTKVIDL; QPMVAAAVI; VPNELNSNL TVRDIPHWL; or TPFPMSCDL.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: ISRDRGWVV; TQKCKVLML; SSLRRRLIL; SLRRRLILA; FAKSSEEEL; PWDKEALM; AVSGKNISL; VCRTKVIDL; DTKDQSHDL; VLSCRNNPL; SCRNNPLII ; QLKGNNSLL; NKHKVGSF; or ECRIKCEDS.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: ISRDRGWVV; TQKCKVLML; SSLRRRLIL; SLRRRLILA; FAKSSEEEL; PWDKEALM; AVSGKNISL; VCRTKVIDL; DTKDQSHDL; VLSCRNNPL; SCRNNPLII ; QLKGNNSLL; NKHKVGSF; or ECRIKCEDS.
  • said polypeptide comprises or consists of a
  • CTL peptide epitope selected from the group: EEPSPPSRA; ADLELPRDA;
  • VEFSNAHGF LEPPLCGQT; VDFQHHQLA; AEAFKQYNI; WELDVLEVS; GECCDPEIT;
  • QETISVQLL QETISVQLL; AEQSCVHFA; CEKTDCPEL; QEMQGQCSV; or NELNSNLKL.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: EQLRLRADL; DAFTLQVWL; TQKCKVLML; AHTALPQLL; KQYNISWEL; SNSSLRRRL; DVNELKNIL; DGHFFEREL; LELEFLYPL; KFVDMDLNL;VCRTKVIDL; AAAVIVHLV; KQETISVQL;
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: SPAVITGLY; SYLPGQWVY; YDGQFMKLY; DNTEVIASY; VRYRVVNLY; GVCDMDCNY; DPDSPHRAY; IGHSLGLY; TPYNNFMSY; LEGRILVQY; CYLELEFLY; DLNLGSVY; YPCTISYPY; SIPDHHQVY; or CEPFMGDNY.
  • a CTL peptide epitope selected from the group: SPAVITGLY; SYLPGQWVY; YDGQFMKLY; DNTEVIASY; VRYRVVNLY; GVCDMDCNY; DPDSPHRAY; IGHSLGLY; TPYNNFMSY; LEGRILVQY; CYLELEFLY; DLNLGSVY; YPCTISYPY; SIPDHHQVY; or CEPFMGDNY
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group: KDPRYFFSL; AEAFKQYNI; NELKNILKL; VLNPSFYGM; REAEGHPDV; VEQPCKSSV; SCRNNPLII; QLKGNNSLL; or QEMQGQCSV.
  • said polypeptide comprises or consists of a CTL peptide epitope selected from the group consisting of
  • said CD40 adjuvant monoclonal antibody is an isotype selected from the group consisting of: IgA, IgM, IgD, IgE and IgG.
  • said isotype is selected from the group consisting of: lgG1 , lgG2, lgG3 and lgG4.
  • Antibodies or immunoglobulins are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain ( ⁇ or ⁇ ), and one pair of heavy (H) chains ( ⁇ , ⁇ , ⁇ , ⁇ and ⁇ ), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant.
  • the L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region.
  • the H chains of Ig molecules are of several classes, ⁇ , ⁇ , ⁇ , a, and ⁇ (of which there are several sub-classes).
  • An assembled Ig molecule consisting of one or more units of two identical H and L chains, derives its name from the H chain that it possesses.
  • Ig isotypes IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., lgG1 , lgG2, lgG3 and lgG4).
  • Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press.
  • said CD40 adjuvant binding fragment is a fragment selected from the group consisting of: Fab, Fab 2 , F(ab') 2 , Fv, Fc, Fd, scFvs.
  • a Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen.
  • Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule.
  • a Fab 2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F (ab') 2 fragment results.
  • An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen.
  • a fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent No 6,248,516.
  • Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions.
  • immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. Immunobiology (cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above.
  • said CD40 adjuvant binding fragment is a single chain antibody fragment.
  • said antibody is a polyclonal antibody.
  • said antibody is a monoclonal antibody.
  • said vaccine or immunogenic composition comprises an adjuvant that is selected from the group consisting of: cytokines selected from the group consisting of GMCSF, interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin 2, interleukin 1 , TGF, TNFa, and TNF3.
  • said adjuvant is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly l:C and derivatives thereof.
  • said adjuvant is a CpG oligonucleotide.
  • said adjuvant is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and/or trehelose dycorynemycolate (TDM).
  • An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, Freunds adjuvant, muramyl dipeptides, liposomes. An adjuvant is therefore an immunomodulator.
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • the term carrier is construed in the following manner.
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid.
  • Such antigens contain B-cell epitopes but no T cell epitopes.
  • the protein moiety of such a conjugate (the "carrier” protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen.
  • Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.
  • T-cell epitopes such as polymers with a repeating B-cell epitope (e.g. bacterial polysaccharides), are intrinsically immunogenic to a limited extent. These are known as T-independent antigens. Such antigens benefit from association with a carrier such as tetanus toxoid, under which circumstance they elicit much stronger antibody responses.
  • said composition comprises a CD40 adjuvant monoclonal antibody, or CD40 adjuvant antibody binding fragment thereof, linked to said pappalysin or pappalysin fragment and a second adjuvant wherein said second different adjuvant as herein disclosed.
  • composition according to the invention for use in the production of antibodies that specifically bind a peptide antigen according to the invention.
  • a composition according to the invention for use in the production of hybridomas that produce monoclonal antibodies that specifically bind a peptide antigen according to the invention are provided.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by uncontrolled cell proliferation.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • cancer includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, haemopoietic system as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • organ systems such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, haemopoietic system as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • said cancer is prostate cancer.
  • said cancer is metastatic prostate cancer of the lymph node or bone.
  • a method to vaccinate a subject suffering from or having a predisposition to cancer comprising administering an effective amount of a vaccine composition according to the invention.
  • said cancer is prostate cancer.
  • said cancer metastatic prostate cancer lymph node or bone In a preferred method of the invention said cancer metastatic prostate cancer lymph node or bone.
  • an ex vivo method for the activation of cytotoxic T cells [CTLs] comprising:
  • said HLA is selected from the group consisting of: HLA- A, HLA-B or HLA-C.
  • said activated CTLs are administered to a patient diagnosed with cancer.
  • said cancer is prostate cancer; preferably metastatic prostate cancer.
  • the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
  • Figure 1 is the amino acid sequence of human pappalysin
  • Figure 2 is the mapping of pappalysin fragments to the domain structure of pappalysin
  • Figure 3 is the amino acid sequence of human pappalysin fragment 1
  • Figure 4 is the amino acid sequence of human pappalysin fragment 2
  • Figure 5 is the amino acid sequence of human pappalysin fragment 3
  • Figure 6 is the amino acid sequence of human pappalysin fragment 4;
  • Figure 7 is the amino acid sequence of human pappalysin fragment 5;
  • Figure 8 is the amino acid sequence of human pappalysin fragment 6;
  • Figure 9 is the effect on tumour growth of ADX-peptide A [AQVATSGEQVGGIFSPLTQKC] ;
  • Figure 10 Expression of pappalysin 1 in tumour RNA by RT-PCR.
  • RT-PCR was used to amplify a fragment corresponding to the protease domain of pappalysin in tumour RNA recovered from immunised and unimmunised mice (arrowhead, top panel).
  • a pappalysin 1 expression plasmid was used as a positive control for pappalysin 1 .
  • GAPDH was amplified as a control for cDNA integrity;
  • Figure 1 Expression of pappalysin 1 in tumour DNA by PCR.
  • RT-PCR was used to amplify a fragment corresponding to the protease domain of pappalysin in total genomic DNA from tumours in immunised and unimmunised mice (arrowhead).
  • a pappalysin 1 expression plasmid was amplified as a positive control.
  • Figure 12 shows tumour growth in the experiment shown in figure 9, but only in those mice in which visible tumours appeared.
  • Figure 13 shows growth of B16 melanoma tumour cells stably transfected with full length murine pappalysin,
  • Figure 14 shows mean tumour size and survival compared between groups immunized with fragment 4 from murine pappalysin
  • Figure 15 shows tumour growth from those groups common to both figures 13 and 14 combined.
  • Figure 16 shows tumour growth as per Figure 15 in only those mice in which visible tumours appeared.
  • Antigenic peptide sequences were derived from the pappalysin protein sequence (figure 1 ) using a proprietary algorithm which computes moving average scores. The score given to a particular amino acid residue reflects both its own properties and the surrounding sequence context. A threshold value is set and while the score remains above the threshold the peptide sequence is elongated. If an amino acid with a score lower than the threshold is encountered then elongation stops and the peptide sequence is defined. If the peptide sequence is more than 10 amino acids long then an overall score for the peptide is calculated.
  • Primers were designed to amplify products approximately corresponding to the predicted protein domains of human PAPPA (see figure 2 and table 1 ). Each forward and reverse primer also contained a 15bp sequence homologous to the BamH 1 site of the His- tagged protein expression vector pET-22b(+) for use in the In-Fusion cloning system (Clontech - see below). PCR was carried using KOD Hot Start DNA polymerase (Novagen) using the following conditions: 95°C for 2mins followed 25 cycles of 95°C 10secs, 55°C 10secs, 70°C 15secs. Products were run on 1 % agarose gel containing 1/10,000 dilution of GelRed (Invitrogen).
  • Cells were pelleted by centrifugation, resuspended in a wash buffer (Tris HCI 50mM; EDTA 2mM, NaCI 50mM pH 7.9) and pelleted once more. Dry pellets were stored at -80 °C until purification.
  • a wash buffer Tris HCI 50mM; EDTA 2mM, NaCI 50mM pH 7.9
  • the resulting pellet was resuspended in a guanidine Isyis buffer. Initially the pellet was resuspended in 5ml resuspension buffer (sodium dihydrogen orthophosphate 20mM; NaCI 0.5M pH 7.8) and 15ml of guanidine lysis buffer added (sodium dihydrogen orthophosphate 20mM; NaCI 0.5M, guanidine HCI 8M ph7.8) resulting in a final concentration of guanidine HCI of 6M.
  • the solubilised protein was incubated at room temperature on a rotating shaker for 10 minutes followed by filtration through a ⁇ . ⁇ syringe filter.
  • a linear elution was carried out by exchanging the native wash buffer with a native elution buffer (sodium dihydrogen orthophosphate 25mM; NaCI 0.5M, imidazole 500mM pH 8) over 15 minutes such that a gradient of 5mM to 500mM imididazole was created over time. 1 ml fractions were collected from the elution at 1 minute intervals.
  • a native elution buffer sodium dihydrogen orthophosphate 25mM; NaCI 0.5M, imidazole 500mM pH 8
  • fractions with high expression were selected for buffer exchange into PBS and further concentration. Fractions were pooled and placed in a Vivaspin 20. PBS was added to make the volume up to 20ml followed by centrifugation at 4000 rpm until the volume was reduced to 5ml. PBS was added to 20ml and the process repeated twice more. Finally the he volume was further reduced to 1 ml. Protein concentration was quantified using a Nanodrop spectrophotometer.
  • B16 mouse melanoma cells were maintained in R10 growth medium which is comprised of RPMI1640 medium supplemented with 10% foetal calf serum (PAA Laboratories Ltd. Yeovil, UK) and 1 % L-Glutamine (Invitrogen, Paisley, UK).
  • B16 cells were plated in 25cm 2 flasks at 5 x 10 5 cells/flask and incubated at 37°C in R10 growth medium for 24h prior to transfection.
  • Cells were transfected with 6 ⁇ g/flask of the pLNCX-PAPPA expression vector using Oligofectamine liposome transfection reagent according to the manufacturer's instructions (Invitrogen, Paisley, UK). Briefly, DNA was mixed with OptiMEM transfection medium (Invitrogen, Paisley, UK).
  • OptiMEM transfection medium In order to select stable transfectants growth media was changed to R10 + 600 ⁇ g/ml G418 72h after transfection. Selection was maintained for 10-14 days to allow the growth of G418 resistant colonies. The cells were then re-plated at one cell/well in 96-well tissue culture plates and maintained in R10 + 600 ⁇ g/ml G418.
  • Buffer exchange PAPPALYSIN antigens into PBS either by dialysis or using an Amicon Ultra-4 spinfilter - 12 ml total (3 spins with 4 ml PBS) is usually sufficient; make sure not to over-concentrate the protein to avoid aggregation Resuspend final retentate at 1 -4 mg/ml in PBS.
  • mice were immunised subcutaneously in the left flank with 10 ⁇ 9 of AQVATSGEQVGGIFSPLTQKC-CD40 peptide conjugate in PBS.
  • Tumour challenge Mice were challenged subcutaneously with 5 x 10 5 B16 melanoma cells which had been transfected with full length murine Pappalysin 14 days earlier. Tumour volume was monitored for up to 28 days until tumours reached 15mm in diameter.
  • RNAIater Sigma
  • DNA and RNA were extracted from excised tumour samples using the AllPrep kit according to the manufacturer's instructions (Qiagen, Crawley, UK).
  • Tumour RNA (500ng) was reverse transcribed into cDNA using random primers and Superscript III reverse transcriptase according to the manufacturer's instructions (Invitrogen, Paisley, UK). Specific primers were used to detect expression of pappalysin (Forward strand 5'-TTGGATGGATCAACACATCTCAATAT-3', reverse strand 5'- CATGGCAGCATCGATCTCCAGGT-3'). Each PCR reaction contained 1 ⁇ of respective forward and reverse primers, 1 .5mM MgCI 2 , 0.2mM dNTPs and 1 U Taq polymerase (GoTaq, Promega).
  • PCR conditions were 94°C for 30s, 35 cycles of 94°C for 20s, 55°C for 10s and 68°C for 1 min followed by 68°C for 7min.
  • GAPDH was also amplified using specific primers with the same PCR conditions as control for the integrity of the cDNA.
  • PCR reaction contained 1 ⁇ of respective forward and reverse primers, 1 .5mM MgCI 2 , 0.2mM dNTPs and 1 U Taq polymerase (GoTaq, Promega). PCR conditions were 94°C for 30s, 35 cycles of 94°C for 20s, 55°C for 10s and 68°C for 1 min followed by 68°C for 7min.
  • Figure 9 shows growth of B16 melanoma tumour cells transiently transfected with full length murine Pappalysin, in unimmunised mice (WT), versus mice immunised a single time subcutaneously with ⁇ g peptide A-ADX40 conjugate.
  • Mice were challenged with 5 x 10 5 B16 cells (subcutaneously, opposite flank) 17 days after immunisation.
  • Figure 10 shows expression of murine Pappalysin mRNA in resected tumours as identified by RT-PCR.
  • Figure 1 1 shows Pappalysin encoding cDNA in tumours as assessed by PCR. 10 mice per group.
  • Figure 12 shows tumour growth in the experiment shown in figure 9, but only in those mice in which visible tumours appeared. A single immunisation with peptide A-ADX40 conjugate was able to slow tumour growth.
  • mice Groups of 10 C57/BI6 female mice (6-10 weeks old, Harlan UK) were immunized subcutaneously (sc) in the left flank on day -28 and day -13 pre-challenge. Two weeks after boosting (day 0) mice received a lethal dose of 5 x 10 5 stably transfected B16mPTT273 cells.
  • sc subcutaneously
  • days 0 Two weeks after boosting (day 0) mice received a lethal dose of 5 x 10 5 stably transfected B16mPTT273 cells.
  • UK Co-ordinating Committee on Cancer Research UK Co-ordinating Committee on Cancer Research (UKCCCR) guidelines that stated tumours larger than 15 mm in diameter had to be culled. New guidelines published in 2010 stated that 12 mm in diameter is the maximum tumour size allowed and therefore challenges 3, 4 and 5 reflect this earlier cut off point.
  • Tumour size was measured using callipers at intervals ranging from every three days to daily for a maximum period of 40 days. Mean tumour
  • Figure 13 shows growth of B16 melanoma tumour cells stably transfected with full length murine Pappalysin, in mice immunised as follows:
  • ADX40 peptide A conjugate black dashed line
  • ADX40 Fragment 2 conjugate Light grey solid line
  • ADX40 Fragment 3 conjugate Light grey dashed line
  • ADX40 Fragment 4 conjugate Dark grey dashed line
  • Example 3 Groups of 10 C57/BI6 female mice (6-10 weeks old, Harlan UK) were immunized subcutaneously (sc) in the left flank on day -28 and day -13 pre-challenge. Two weeks after boosting (day 0) mice received a lethal dose of 5 x 10 5 stably transfected B16mPTT273 cells.
  • sc subcutaneously
  • day 0 mice received a lethal dose of 5 x 10 5 stably transfected B16mPTT273 cells.
  • Early work was based on UK Co-ordinating Committee on Cancer Research (UKCCCR) guidelines that stated tumours larger than 15 mm in diameter had to be culled. New guidelines published in 2010 stated that 12 mm in diameter is the maximum tumour size allowed and therefore these data reflect this earlier cut off point. Tumour size was measured using callipers at intervals ranging from every three days to daily for a maximum period of 40 days. Mean tumour size and survival were compared between groups and are shown in figure 14.
  • mice were immunised as follows:
  • Fragment 4 (Fr4): light grey solid line
  • ADX40-Fragment 4 conjugate black dashed line

Abstract

L'invention concerne une composition vaccinale comprenant des fragments et des peptides dérivés d'un polypeptide pappalsyin, et l'utilisation du vaccin pour la prévention ou le traitement du cancer.
PCT/GB2011/051096 2010-06-16 2011-06-13 Vaccin à base de polypeptides WO2011158019A1 (fr)

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GBGB1010054.3A GB201010054D0 (en) 2010-06-16 2010-06-16 CTL vaccine
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GBGB1010058.4A GB201010058D0 (en) 2010-06-16 2010-06-16 Peptide vaccine
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GBGB1010056.8A GB201010056D0 (en) 2010-06-16 2010-06-16 Polypeptide vaccine

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