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Title:
POLYPEPTIDE VACCINE
Document Type and Number:
WIPO Patent Application WO/2011/158019
Kind Code:
A1
Abstract:
We disclose a vaccine composition comprising fragments and peptides derived from a pappalsyin polypeptide and the use of the vaccine in the prevention and treatment of cancer.

Inventors:
CARLING-WRIGHT, Jennifer (Adjuvantix Limited, Innovation Centre217 Portobello,Sheffield, Yorkshire S1 4DP, GB)
BIRNIE, Richard (Procure Therapeutics Limited, BiocentreInnovation Way,York, Yorkshire YO10 5DG, GB)
HEATH, Andrew William (Adjuvantix Limited, Innovation Centre217 Portobello,Sheffield, Yorkshire S1 4DP, GB)
MAITLAND, Norman (Procure Therapeutics Limited, BiocentreInnovation Way,York, Yorkshire YO10 5DG, GB)
Application Number:
GB2011/051096
Publication Date:
December 22, 2011
Filing Date:
June 13, 2011
Export Citation:
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Assignee:
ADJUVANTIX LIMITED (Innovation Centre, 217 PortobelloSheffield, Yorkshire S1 4DP, GB)
PROCURE THERAPEUTICS LIMITED (Biocentre, Innovation WayYork, Yorkshire YO10 5DG, GB)
CARLING-WRIGHT, Jennifer (Adjuvantix Limited, Innovation Centre217 Portobello,Sheffield, Yorkshire S1 4DP, GB)
BIRNIE, Richard (Procure Therapeutics Limited, BiocentreInnovation Way,York, Yorkshire YO10 5DG, GB)
HEATH, Andrew William (Adjuvantix Limited, Innovation Centre217 Portobello,Sheffield, Yorkshire S1 4DP, GB)
MAITLAND, Norman (Procure Therapeutics Limited, BiocentreInnovation Way,York, Yorkshire YO10 5DG, GB)
International Classes:
A61K39/00; C07K16/30; A61P35/00
Attorney, Agent or Firm:
SYMBIOSIS IP (Symbiosis IP Limited, Apollo HouseEboracum Way,Heworth Green, York Yorkshire YO31 7RE, GB)
Download PDF:
Claims:
Claims

1 A vaccine or immunogenic composition wherein said composition comprises:

iii) a CD40 adjuvant monoclonal antibody, or CD40 adjuvant antibody binding fragment thereof, linked to a pappalysin, or pappalysin polypeptide fragment; or

iv) a pappalysin peptide fragment derived from pappalysin and an adjuvant wherein said adjuvant is not a CD40 monoclonal antibody, or CD40 antibody fragment.

2 The composition according to claim 1 wherein said pappalysin is human pappalysin 1 .

3. The composition wherein said pappalysin is represented by the amino acid sequence in Figure 1 .

4. The composition according to any of claims 1 -3 wherein said pappalysin polypeptide fragment is a peptide. 5. The composition according to claim 4 wherein said peptide is 10-30 amino acids in length.

6. The composition according to claim 4 wherein said peptide is 10-18 amino acids in length.

7. The composition according to claim 4 wherein said peptide is 10 amino acids in length.

8. The composition according to claim 4 wherein said peptide is 29 amino acids in length.

9. The composition according to any of claims 1 -8 wherein 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.

10. The composition according to claim 9 wherein said fragment comprises or consists of one or more peptides selected from the group consisting of:

ERPRRARRDPRAGRPPRPAAG;

VRVPRRRQQREARGATEEPSPPSR;

TISDQDNKDPRYF;

iv) RAARGRRASPPPPPPPGG;

v) FSGRGEQLRVLRADLELPRDAF;

vi) LKTDRARQVT;

vii) VARTQREILSDMETHGA;

viii) LYDKCSYISRDRGW;

ix) SALNHNYRGY; or

x) NAHRSYLPGQ, wherein said fragment includes

more pappalysin epitopes.

1 1 . The composition according to any of claims 1 -8 wherein 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. 12. The composition according to claim 1 1 wherein said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) LYEDDHKNPTVTREQVD;

ϋ) KKQHNGVCDMDCNYERFNFDGGECC;

v) DISKIGDENCDPECN;

iv) SYNQLSSFRQPKVVRYR;

vi) CFDPDSPHRA;

vii) EVSNSSLRRRLI;

vii) TGHDGGDCRHLRH; or

v) EAFKQYNISW, wherein said fragment includes one or more pappalysin epitopes.

13. The composition according to any of claims 1 -8 wherein 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.

14. The composition according to claim 13 wherein said fragment comprises consists of one or more peptides selected from the group consisting of:

i) EIQSCSDPCMETEPSFETGDLCNDT;

SSPMPCSPSGHWSPREAEGHPDVE;

SSPMPCSPSGHWSPREAEGHPDVE;

iv) QGWQPSRKPA;

v) CGDPGPGNDT; or

vi) SVRTWSPNSAVNPHTV, wherein said fragment includes one or more pappalysin epitopes.

15. The composition according to any of claims 1 -8 wherein 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. 16. The composition according to claim 15 wherein said fragment comprises or consists of one or more peptides selected from the group consisting of:

GRGYCGDGIIQKDQGEQCDDMNKINGDGC;

HDGDGVCEEFEQKTSIKDCG;

SSCQRGETYSPAEQSC;

iv) VTDGTYYGDQKQETI;

v) LLDTKDQSHDL;

vi) CKPLKYKVVRDPPLQMD;

vii) SGTEESEPSPA;

viii) QVCRTKVIDLSEGISQHA;

ix) WASNASVSHQDQQCP;

x) QEVSFNCIDEPSR; or xi) RSFDNFDPVT, wherein said fragment includes one or more pappalysin epitopes.

17. The composition according to any of claims 1 -8 wherein 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.

18. The composition according to claim 17 wherein said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) VPGSSRKSKKRAFKTQCTQDGSWQEGA;

ii) PPVPNADLQTARCRENKHKVGS;

iii) FNSECRIKCEDSDASQGLG;

iv) QIRRDDELIKSQTGPSV;

v) EMQGQCSVPNELNSNLKLQCPDG;

vi) NASLNCSSSDRYHG;

vii) FQCRHPAQLKGNNS;

viii) HCRKDGTWNGS;

ix) ACEKTDCPELAV;

x) EPVDCSIPDHHQ; or

xi) CPEGTTFGSQC, wherein said fragment includes one or more pappalysin epitopes.

19. The composition according to any of claims 1 -8 wherein 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.

20. The composition according to claim 19 wherein said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) DLQGDCACRDPQAQEHSRKDLRGYSHG;

ii) WLNPTRVERV; or iii) FMGDNYCDAINNRA, wherein said fragment includes one or more pappalysin epitopes.

21 . The composition according to any of claims 1 -8 wherein said peptide comprises or consists of the amino acid sequence:

i) AQVATSGEQVGGIFSPLTQKC.

or wherein said 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.

22. The composition according to any of claims 1 -8 wherein 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.

23. The composition according to any of claims 1 -8 wherein 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 GTWNGSFHV.

24. The composition according to any of claims 1 -8 wherein said polypeptide comprises or consists of a CTL peptide epitope selected from the group: AVITGLYDK; ATYDGQFMK; GIFSPLTQK; QLSSFRQPK; ILANCDISK; HLRHPAFVK; GVATWPWDK; GVCEEFEQK; or GLYQCTNGF.

25. The composition according to any of claims 1 -8 wherein 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.

26. The composition according to any of claims 1 -8 wherein 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.

27. The composition according to any of claims 1 -8 wherein said polypeptide comprises or consists of a CTL peptide epitope selected from the group: EEPSPPSRA;

ADLELPRDA; RDAFTLQVW; AEGGQRSPA; GEQVGGIFS; REILSDMET; METHGAHTA; VEFSNAHGF; LEPPLCGQT; VDFQHHQLA; AEAFKQYNI; WELDVLEVS; GECCDPEIT; NELKNILKL; SEEELAGVA; EELAGVATW; HEIGHSLGL; METEPSFET; RELGSACHL; REAEGHPDV; VEQPCKSSV; LELEFLYPL; CDVPLTIRL; EEVYGIQIY; DEHLEIDAA; LEIDAAMLT; MDVASILHL; TEESEPSPA; SEPSPAVTY; EEFEQKTSI; SEGISQHAW; QETISVQLL; AEQSCVHFA; CEKTDCPEL; QEMQGQCSV; or NELNSNLKL.

28. The composition according to any of claims 1 -8 wherein 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; VRVSFSSPL;VPNELNSNL; or TPFPMSCDL. 29. The composition according to any of claims 1 -8 wherein 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.

30. The composition according to any of claims 1 -8 wherein said polypeptide comprises or consists of a CTL peptide epitope selected from the group: KDPRYFFSL; AEAFKQYNI; NELKNILKL; VLNPSFYGM; REAEGHPDV; VEQPCKSSV; SCRNNPLII; QLKGNNSLL; or QEMQGQCSV.

31 . The composition according to any of claims 1 -30 wherein said CD40 adjuvant monoclonal antibody is an isotype selected from the group consisting of: IgA, IgM, IgD, IgE and IgG.

32. The composition according to claim 31 wherein said isotype is selected from the group consisting of: lgG1 , lgG2, lgG3 and lgG4.

33. The composition according to any of claims 1 -30 wherein said CD40 adjuvant binding fragment is selected from the group consisting of: Fab, Fab2, F(ab')2, Fv, Fc, Fd, scFvs.

34. The composition according to claim 33 wherein said CD40 binding fragment is a single chain antibody fragment [scFvs].

35. The composition according to any one of claims 1 -34 wherein 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.

36. The composition according to any of claims 1 -35 for use in the production of antibodies that specifically bind a fragment or peptide antigen as herein disclosed.

37. The composition according to claim 36 wherein said antibody is a polyclonal antibody. 38. The composition according to claim 36 wherein said antibody is a monoclonal antibody.

39. The composition according to any of claims 1 -21 for use in the production of a hybridoma that produces a monoclonal antibody that specifically bind a fragment or peptide antigen as hereindisclosed.

40. A vaccine according to any of claims 1 -38 for use in the treatment of cancer.

41 . The vaccine according to claim 40 wherein said cancer is prostate cancer.

42. The vaccine according to claim 41 wherein said cancer is metastatic prostate cancer of the lymph node or bone.

43. 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 any of claims 1 -38.

44. The method according to claim 43 wherein said cancer is prostate cancer.

45. The method according to claim 44 wherein said cancer is metastatic prostate cancer of the lymph node or bone.

46. An ex vivo method for the activation of cytotoxic T cells [CTLs] comprising:

i) obtaining a sample comprising CTLs from a subject;

ii) contacting the sample with at least one peptide antigen according to any one of claims 22 to 30 wherein said antigen is presented to the CTL via a type 1 HLA molecule to form an activation culture;

iii) incubating the culture formed in ii) to expand the activated CTL specific for said peptide antigen; and optionally

iv) collecting said activated CTLs.

47. The method according to claim 46 wherein said HLA is selected from the group consisting of: HLA- A, HLA-B or HLA-C. 48. The method according to claim 46 or 47 wherein said activated CTLs are administered to a patient diagnosed with cancer.

49. The method according to any one of claims 46 to 48 wherein said cancer is prostate cancer.

50. The method according to claim 49 wherein prostate cancer is metastatic prostate cancer.

Description:
Polypeptide Vaccine

We disclose 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.

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. Some 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. In addition to its importance in T and B-cell interactions, 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. It is known that ligation of CD40 by adjuvant antibodies can effectively replace the CD40 signals ordinarily made during intercellular interaction in the immune response (see WO03/063899; WO2004/052396 and WO2004/041866). The development of subunit vaccines has been the focus of considerable research in recent years. The discovery of novel vaccine antigens from genomic and proteomic studies is enabling the development of new subunit vaccine candidates, particularly against cancers. However, although subunit vaccines have advantages of specificity their 'pure' status means that subunit vaccines do not always have adequate immunogenicity. Many candidate subunit vaccines have failed in clinical trials in recent years that might otherwise have succeeded were a suitable adjuvant available to enhance the immune response to the purified antigen.

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]. According to an aspect of the invention there is provided a vaccine or immunogenic composition comprising:

i) a pappalysin or a polypeptide fragment derived from a pappalysin;

ii) a CD40 monoclonal antibody, or CD40 antibody binding fragment

thereof, linked to said pappalysin or pappalysin fragment.

In a preferred embodiment of the invention there is provided a pappalysin peptide fragment derived from a pappalysin and an adjuvant wherein said adjuvant is not a CD40 adjuvant monoclonal antibody, or CD40 adjuvant antibody fragment.

According to an aspect of the invention there is provided a vaccine or immunogenic composition wherein said composition comprises:

i) a CD40 adjuvant monoclonal antibody, or CD40 adjuvant antibody binding fragment thereof, linked to a pappalysin, or pappalysin polypeptide fragment; or

ii) a pappalysin peptide fragment derived from pappalysin and an adjuvant wherein said adjuvant is not a CD40 monoclonal antibody, or CD40 antibody fragment.

In an alternative aspect of the invention there is provided 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..

In a further alternative preferred aspect of the invention there is provided 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.

In a preferred embodiment of the invention said pappalysin is human pappalysin 1 . Preferably said pappalysin is represented by the amino acid sequence in Figure 1 .

In a preferred embodiment of the invention said fragment is a peptide. In a preferred embodiment of the invention said peptide is 10-30 amino acids in length; preferably said peptide is 8-18 amino acids in length.

In a preferred embodiment of the invention said peptide is 10 amino acids in length.

In an alternative preferred embodiment of the invention said peptide is 29 amino acids in length.

In a preferred embodiment of the invention 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. In a preferred embodiment of the invention said fragment comprises or consists of one or more peptides selected from the group consisting of:

ERPRRARRDPRAGRPPRPAAG;

VRVPRRRQQREARGATEEPSPPSR;

TISDQDNKDPRYF;

iv) RAARGRRASPPPPPPPGG;

v) FSGRGEQLRVLRADLELPRDAF;

vi) LKTDRARQVT;

vii) VARTQREILSDMETHGA;

viii) LYDKCSYISRDRGW;

ix) SALNHNYRGY; or

x) NAHRSYLPGQ, wherein said fragment includes pappalysin epitopes.

In a preferred embodiment of the invention 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.

In a preferred embodiment of the invention said fragment comprises or consists of one or more peptides selected from the group consisting of: LYEDDHKNPTVTREQVD;

VKKQHNGVCDMDCNYERFNFDGGECC;

DISKIGDENCDPECN;

iv) SYNQLSSFRQPKVVRYR;

v) CFDPDSPHRA;

vi) EVSNSSLRRRLI;

vii) TGHDGGDCRHLRH; or

viii) EAFKQYNISW, wherein said fragment includes

more pappalysin epitopes.

In a preferred embodiment of the invention 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.

In a preferred embodiment of the invention said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) EIQSCSDPCMETEPSFETGDLCNDT;

ii) SSPMPCSPSGHWSPREAEGHPDVE;

iii) QGWQPSRKPA;

iv) CGDPGPGNDT; or

ix) SVRTWSPNSAVNPHTV, wherein said fragment includes one or more pappalysin epitopes.

In a preferred embodiment of the invention 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.

In a preferred embodiment of the invention said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) GRGYCGDGIIQKDQGEQCDDMNKINGDGC;

ϋ) HDGDGVCEEFEQKTSIKDCG;

iii) SSCQRGETYSPAEQSC; iv) VTDGTYYGDQKQETI;

v) LLDTKDQSHDL;

vi) CKPLKYKVVRDPPLQMD;

vii) SGTEESEPSPA;

viii) QVCRTKVIDLSEGISQHA;

ix) WASNASVSHQDQQCP;

x) QEVSFNCIDEPSR; or

x) RSFDNFDPVT, wherein said fragment includes one or more pappalysin epitopes.

In a preferred embodiment of the invention 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.

In a preferred embodiment of the invention said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) VPGSSRKSKKRAFKTQCTQDGSWQEGA;

ϋ) PPVPNADLQTARCRENKHKVGS;

iii) FNSECRIKCEDSDASQGLG;

iv) QIRRDDELIKSQTGPSV;

v) EMQGQCSVPNELNSNLKLQCPDG;

vi) NASLNCSSSDRYHG;

vii) FQCRHPAQLKGNNS;

viii) HCRKDGTWNGS;

ix) ACEKTDCPELAV;

x) EPVDCSIPDHHQ; or

xi) CPEGTTFGSQC, wherein said fragment includes one or more pappalysin epitopes.

In a preferred embodiment of the invention 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. In a preferred embodiment of the invention said fragment comprises or consists of one or more peptides selected from the group consisting of:

i) DLQGDCACRDPQAQEHSRKDLRGYSHG;

ii) WLNPTRVERV; or

iii) FMGDNYCDAINNRA, wherein said fragment includes one or more pappalysin epitopes.

In a preferred embodiment of the invention said peptide comprises or consists of the amino acid sequence:

i) AQVATSGEQVGGIFSPLTQKC; or

ii) AQVATSGEQVGGIFSPLTQKC or wherein said 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.

In a preferred embodiment of the invention 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.

In a preferred embodiment of the invention 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. In a preferred embodiment of the invention said polypeptide comprises or consists of a CTL peptide epitope selected from the group: AVITGLYDK; ATYDGQFMK; GIFSPLTQK; QLSSFRQPK; ILANCDISK; HLRHPAFVK; GVATWPWDK; GVCEEFEQK; or GLYQCTNGF. In a preferred embodiment of the invention 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. In a preferred embodiment of the invention 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. In a preferred embodiment of the invention said polypeptide comprises or consists of a

CTL peptide epitope selected from the group: EEPSPPSRA; ADLELPRDA;

RDAFTLQVW; AEGGQRSPA; GEQVGGIFS; REILSDMET; METHGAHTA;

VEFSNAHGF; LEPPLCGQT; VDFQHHQLA; AEAFKQYNI; WELDVLEVS; GECCDPEIT;

NELKNILKL; SEEELAGVA; EELAGVATW; HEIGHSLGL; METEPSFET; RELGSACHL; REAEGHPDV; VEQPCKSSV; LELEFLYPL; CDVPLTIRL; EEVYGIQIY; DEHLEIDAA;

LEIDAAMLT; MDVASILHL; TEESEPSPA; SEPSPAVTY; EEFEQKTSI; SEGISQHAW;

QETISVQLL; AEQSCVHFA; CEKTDCPEL; QEMQGQCSV; or NELNSNLKL.

In a preferred embodiment of the invention 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;

VRVSFSSPL;VPNELNSNL; or TPFPMSCDL In a preferred embodiment of the invention 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.

In a preferred embodiment of the invention said polypeptide comprises or consists of a CTL peptide epitope selected from the group: KDPRYFFSL; AEAFKQYNI; NELKNILKL; VLNPSFYGM; REAEGHPDV; VEQPCKSSV; SCRNNPLII; QLKGNNSLL; or QEMQGQCSV. In a preferred embodiment of the invention said polypeptide comprises or consists of a CTL peptide epitope selected from the group consisting of

i) AQVATSGEQVGGIFSPLTQKC; or

ii) ATYDGQFMKLYVNGAQVA.

In a preferred embodiment of the invention said CD40 adjuvant monoclonal antibody is an isotype selected from the group consisting of: IgA, IgM, IgD, IgE and IgG.

In a preferred embodiment of the invention said isotype is selected from the group consisting of: lgG1 , lgG2, lgG3 and lgG4.

Antibodies or immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (κ or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), 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, α, μ, σ, a, and γ (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. Thus, there are five 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.

In a preferred embodiment of the invention said CD40 adjuvant binding fragment is a fragment selected from the group consisting of: Fab, Fab 2 , F(ab') 2 , Fv, Fc, Fd, scFvs.

Various fragments of antibodies are known in the art, e.g. Fab, Fab 2 , F(ab') 2 , Fv, Fc, Fd, scFvs, etc. 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. These and other 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.

In a preferred embodiment of the invention said CD40 adjuvant binding fragment is a single chain antibody fragment.

In a preferred embodiment of the invention said antibody is a polyclonal antibody.

In an alternative preferred embodiment of the invention said antibody is a monoclonal antibody.

In an alternative preferred embodiment of the invention 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. In a further alternative embodiment of the invention said adjuvant is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly l:C and derivatives thereof. In a preferred embodiment of the invention said adjuvant is a CpG oligonucleotide.

In a preferred embodiment of the invention 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. Some 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. Certain antigens which lack 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. In a preferred embodiment of the invention 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. According to a further aspect of the invention there is provided a composition according to the invention for use in the production of antibodies that specifically bind a peptide antigen according to the invention. According to a further aspect of the invention there is provided 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.

The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, "Basic Facts about Hybridomas" in Compendium of Immunology V.ll ed. by Schwartz, 1981 , which are incorporated by reference. According to a further aspect of the invention there is provided at least one peptide antigen according to the invention for use in the treatment of cancer.

As used herein, the term "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. The term "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. The term "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. The term "carcinoma" 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. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.

In a preferred embodiment of the invention said cancer is prostate cancer.

In a preferred embodiment of the invention said cancer is metastatic prostate cancer of the lymph node or bone.

According to a further aspect of the invention there is provided 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.

In a preferred method of the invention said cancer is prostate cancer.

In a preferred method of the invention said cancer metastatic prostate cancer lymph node or bone.

According to a further aspect of the invention there is provided an ex vivo method for the activation of cytotoxic T cells [CTLs] comprising:

i) obtaining a sample comprising CTLs from a subject;

ii) contacting the sample with at least one peptide antigen according to the invention wherein said antigen is presented to the CTL via a type 1 HLA molecule to form an activation culture;

iii) incubating the culture formed in ii) to expand the activated CTL specific for said peptide antigen; and optionally

iv) collecting said activated CTLs.

In a preferred method of the invention said HLA is selected from the group consisting of: HLA- A, HLA-B or HLA-C.

In a preferred method of the invention said activated CTLs are administered to a patient diagnosed with cancer.

In a preferred method of the invention said cancer is prostate cancer; preferably metastatic prostate cancer. Throughout the description and claims of this specification, 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.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

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 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; and

Figure 16 shows tumour growth as per Figure 15 in only those mice in which visible tumours appeared.

Materials and Methods Derivation of Pappalysin Peptides

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.

Cloning of mouse PAPPA fragments into pET22b(+) expression vector

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). Bands were visualized using a UV transilluminator (GeneGenius). pET-22b(+) was linearized with BamH1 (37°C, 3h) and products run on a 0.8% agarose gel stained with GelRed. A band corresponding to linearized vector was excised and the DNA purified using a Qiagen Gel Extraction kit following the manufacturer's protocol. Insertion of the fragment DNA into the vector was accomplished using the Clontech In- Fusion Advantage kit following manufacturer's instructions. The resulting constructs were transformed into DH5a competent bacteria followed by culture on Luria broth (LB) agar containing ampicillin (50μg/ml; Sigma). Plates were incubated overnight at 37°C. Individual colonies were picked into 5ml LB containing ampicillin and incubated overnight in a shaker incubator. DNA was extracted using a Qiaprep Spin Miniprep kit (Qiagen) following manufacturer's instructions. DNA sequencing confirmed that the insert was in frame with the His tag required for purification (Technology Facility, University of York) The construct was transformed into Rosetta-gami2 (DE3) pLysS expression hosts. Induction of protein expression

Bulk inductions were carried out using the same conditions as described above. Rosetta-gami2 (DE3) pLysS cells containing the relevant pappalysin fragment were inoculated into 10ml LB with ampicillin (50μg/ml; Sigma) and incubated at 37 < C in a shaker incubator. When the OD 600 reached 0.5, the culture was added to 500ml of LB containing ampicillin. When the OD600 reached 0.5 units 1 mM IPTG was added and the culture incubated for a further 2 hours. 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.

Purification under denaturing conditions

Preliminary experiments showed that the fragments were packaged into insoluble inclusion bodies, therefore, fragments were purified under denaturing conditions. Bacterial cell pellets from 500ml cultures were resuspended in 10ml PBS followed by sonication on ice (Soniprep 150, MSE; 4 x 30 sec. bursts interspersed with 15 sec. cooling). Lysed culture was spun at 10,000xg for 15 minutes. The supernatant was discarded and the pellet of insoluble material was resuspended in 10ml of PBS and centrifuged once more,

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 Ο.δμηι syringe filter.

Purification was carried out using a 1 ml HisTrap column (GE Healthcare) charged with 3% Ni S0 4 attached to a AKTA purifier (Amersham). The solubilised protein was passed over the column at a rate of 1 ml/min with a denaturing binding buffer (sodium dihydrogen orthophosphate 20mM; NaCI 0.5M, Urea 8M pH7.8). The column was washed using a denaturing wash buffer (sodium dihydrogen orthophosphate 20mM; NaCI 0.5M, Urea 8M pH6) which was gradually replaced by native wash buffer (sodium dihydrogen orthophosphate 25mM; NaCI 0.5M, imidazole 5mM pH 8) over a 30 mins period. 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.

Buffer exchange and concentration of protein

After PAGE analysis of the eluted fragments, 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.

Culture of B16 cells

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).

Stable transfection of B16 cells

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). In order to select stable transfectants growth media was changed to R10 + 600μg/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 + 600μg/ml G418.

Conjugation of Pappalvsin fragments to CD40 Mab

Materials

Use fresh Sulfo-SMCC to activate antigens

Use fresh SATA to activate antibodies

Hydroxylamine buffer (50 ml): 0.5 M Hydroxylamine

25 mM EDTA in PBS

pH 7.2-7.5.

Dissolve 1 .74 g hydroxylamine « HCI and EDTA (0.475 g of tetrasodium salt or 0.365 g of disodium salt) in 40 ml PBS. Add ultrapure water to a final volume of 50 ml and adjust pH to 7.2-7.5 with NaOH. Amicon Ultra-4 filters:

- add 3 ml PBS to 30 kD centrifuge filter

- spin at 1400 g for 15 min, RT

- remove any remaining PBS from insert

Buffer exchange PAPPALYSIN antigens

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.

Block -SH groups in PAPPALYSIN antigen

- dissolve N-ethyl-maleimide at 25 mg/ml in dH20

- add equal amount (mg) of NEM to PAPPALYSIN antigen

- incubate for 2 hours at RT on shaker/rotator

Activation of PAPPALYSIN antigens with Sulfo-SMCC

- dissolve 2 mg Sulfo-SMCC in 600 μΙ milliQ; if this does not dissolve easily, warm to 50°C

- add 60 μΙ Sulfo-SMCC solution to 1 ml PAPPALYSIN antigen solution (1 -4 mg/ml)

- incubate for 1 hour at RT on shaker/rotator

- transfer solution to an Amicon Ultra-4 spinfilter

- add up to 4 ml PBS, spin at 1400g for 10-20 min or until retentate ~ 0.5 ml (avoid over- concentrating the protein solution!); repeat 3 times

- resuspend retentate in 0.3 - 0.5 ml PBS.

Activation of anti-CD40 antibody

- dissolve 6-8 mg SATA per 500 μΙ DMSO

- add 10 μΙ SATA-solution to 1 ml anti-CD40 (2-10 mg/ml)

- incubate for 30 minutes at RT - transfer solution to an Amicon Ultra-4 spinfilter

- add up to 4 ml PBS, spin at 1400g for 10-20 min or until retentate ~ 0.5 ml (avoid over- concentrating the protein solution!); repeat 3 times

(At this stage the SATA-treated protein can be stored indefinitely at -20C for later use) Continued protocol:

- resuspend 1 mg antibody at 1 mg/ml

- add 100 μΙ hydroxylamine buffer to 1 mg antibody

- incubate for 2 hours at RT on mixer/rotator. Conjugation

- mix 1 mg maleimide-activated PAPPALYSIN antigen (0.3 - 0.5 ml) with 1 mg sulfhydryl-anti-CD40 (1 .1 ml), or use an appropriate amount of PAPPALYSIN antigen for the desired molar ratio. A molar ratio of 2(PAPPALYSIN):1 (CD40) may work well

- incubate overnight at 4°C on shaker/rotator

- make up a fresh stock solution of 1 M L-cysteine solution in MilliQ

- add L-cysteine to the conjugated proteins at a final concentration of 50 mM in order to stop the reaction

- incubate for 15 min at RT

- transfer solution to an Amicon Ultra-4 spinfilter

- add up to 4 ml PBS, spin at 1400g for 10-20 min or until retentate ~ 0.5 ml (avoid over- concentrating the protein solution !); repeat 3 times

- resuspend conjugate in 1 ml PBS

- store conjugate at 4C; if stored for a prolonged length of time, add 0.01 % sodium azide

Conjugation of Pappalvsin peptides to CD40 mAb

Conjugation of peptides is normally as described above for fragments, but a higher ratio of peptide to antibody would be used. For instance, in conjugating the peptide fragment AQVATSGEQVGGIFSPLTQKC to immunize mice and challenge as shown in fig x, 148pg of peptide was conjugated to lmg of SATA derivatised CD40 antibody, a 10: 1 molar ratio of peptide to CD40 mAb. In addition, as the peptide had a terminal cysteine residue, the peptide was not reacted with SMCC but was conjugated to the SATA derivatised antibody directly through the C- terminal cysteine. Such an approach might be appropriate for any peptide with a cysteine residue, and indeed a cysteine might be added to the natural sequence to facilitate conjugation in this manner. Immunization of Mice

C57/BI6 female mice were immunised subcutaneously in the left flank with 10μ9 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.

Extraction of tumour DNA/RNA from excised tumours

Tumours were resected and placed immediately into RNAIater (Sigma) and stored at - 20°C. DNA and RNA were extracted from excised tumour samples using the AllPrep kit according to the manufacturer's instructions (Qiagen, Crawley, UK).

RT-PCR detection of Pappalysin expression in tumour RNA

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 μΜ 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 detection of Pappalysin DNA in tumour DNA

Specific primers were used to detect the pappalysin expression construct in 500ng of tumour derived total genomic DNA (Forward strand 5'- TTGGATGGATCAACACATCTCAATAT-3', reverse strand 5'- CATGGCAGCATCGATCTCCAGGT-3'). Each PCR reaction contained 1 μΜ 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.

Example 1

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.

Example 2

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. 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 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 size and survival were compared between groups.

Figure 13 shows growth of B16 melanoma tumour cells stably transfected with full length murine Pappalysin, in mice immunised as follows:

PBS: Solid black line

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. 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:

Control PBS: solid black line

Fragment 4 (Fr4): light grey solid line

ADX40: light grey dashed line

ADX40-Fragment 4 conjugate: black dashed line

Figure 15 shows tumour growth from those groups common to both figures 13 and 14 combined. 20 mice per group. N=20 for PBS and ADX40-Fr4, n=10 for Fr4 and ADX40 groups Figure 16 shows tumour growth as above, in only those mice in which visible tumours appeared. 20 mice per group n=18 for PBS and ADX40-Fr4, n=10 for Fr4 and ADX40 groups.