WO2016080830A2 - Immunotherapeutic method for treating pancreatic cancer - Google Patents

Abstract

The invention relates to treatment of pancreatic cancer and metastases thereof. More specifically, the invention relates to immunogenic polypeptides comprising at least a portion of a pancreatic tumor cell-associated protein or immunologically active variants thereof and to nucleic acids encoding such polypeptides and to the use thereof in immunotherapeutic methods of treatment of pancreatic cancer. Said immunogenic polypeptides are provided by the zona pellucida (ZP) (glyco)proteins. ZP (glyco)proteins and fragments thereof that can induce a CD8⁺ and/or CD4⁺ T cell response as well as nucleic acid sequences encoding them can suitably be used in the present immunotherapeutic strategies.

Description

IMMUNOTHERAPEUTIC METHOD FOR TREATING PANCREATIC

CANCER

FIELD OF THE INVENTION

The present invention relates to the field of treatment of pancreatic cancer and metastases thereof. More specifically, the invention relates to immunogenic polypeptides comprising at least a portion of a pancreatic tumour cell associated (glyco)protein or immunologically active variants thereof and to nucleic acids encoding such polypeptides. Such polypeptides and nucleic acid sequences may be used in vaccines and pharmaceutical compositions for therapeutic and prophylactic treatment of pancreatic cancer and metastases thereof.

BACKGROUND OF THE INVENTION

Pancreatic cancer is the fifth leading cause of death by cancer in western society. Patients diagnosed with pancreatic cancer typically have a poor prognosis. The 5-year survival for pancreatic cancer patients is less than 5% and the incidence of the disease has tripled over the last 40 years. With, for example, 37, 170 cases diagnosed in the United States in 2007, and 33,700 deaths, pancreatic cancer has one of the highest fatality rates of all cancers.

Pancreatic tumours may arise from ductal, acinar and stromal cells. 80 % of pancreatic carcinomas are derived from ductal epithelium. 60% of these tumors are located in the head of the pancreas, 10% in the tail and 30% are located in the body of the pancreas or are diffuse. Pancreatic tumours are typically aggressive in terms of metastasis, locally as well as to visceral organs.

The molecular basis underlying the pathogenesis of pancreatic adenocarcinoma remains unknown, which is one of the reasons that the disease lacks early diagnostic and therapeutic modalities.

Because pancreatic tumours usually cause no symptoms early on, it often has progressed to locally advanced or metastatic disease at time of diagnosis, resulting in an extremely poor prognosis.

At present, 'curative' strategies most commonly include surgery and/or chemotherapy. The most common sugical procedure is the pancreaticoduodenectomy (or 'Whipple procedure'), is performed to treat cancerous tumours on the head of the pancreas or cancerous tumors on ducts or vessels near the pancreas. This procedure can only be performed if the patient's condition allows for major surgery and if the cancer is localised without invading local structures or metastases, and therefore proves unfit for the majority of cases. Spleen-preserving distal pancreatectomy is used to remove a cancer running through centre of pancreas; this also constitutes major invasive surgery. Recently, localized cancers of the pancreas have been resected using less invasive (laparoscopic) approaches. After surgery, adjuvant chemotherapy with gemcitabine may be offered to eliminate whatever cancerous tissue may remain in the body. This has been shown to increase 5-year survival rates. The benefit of additional radiation therapy has not been proven and is presently the subject of debate.

In patients not suitable for resection with curative intent, palliative chemotherapy may improve quality of life and gain a modest survival benefit. In these cases Gemcitabine is administered intravenously on a weekly basis. Addition of oxaliplatin (Gem/Ox) conferred benefit in small trials, but is not yet standard therapy. Fluorouracil (5FU) may also be included.

All in all, only 20% of the tumors are resectable and the survival benefit of approved chemotherapy regiments is rather poor. The identification of novel therapeutic targets and agents is therefore of paramount importance for improving the prognosis of pancreatic cancer patients. Recent advances in molecular medicine have increased the general interest in tumour-specific cell surface antigens that could serve as targets for various immunotherapeutic strategies.

Among the various elements of the immune system, T lymphocytes are probably the most adept to recognize and eliminate cells expressing foreign or tumour-associated antigens. Cytotoxic T Lymphocytes (CTLs) express the CD8 cell surface marker and are specialized at inducing lysis of the target cells with which they react via the perforin/granzyme and/or the Fas/Fas-L pathways. The T-cell receptor (TCR) for antigen of CTLs binds to a molecular complex on the surface of the target cell formed by small peptides (8-1 1) residues derived from processed foreign or tumour associated antigens, which associate with major histocompatibility complex (MHC) class I molecules.

The other major T-cell subset, helper T lymphocytes (HTLs or T helper cells), is characterized by the expression of CD4 surface marker. The T helper cells recognize slightly larger peptides (1 1-20 residues), also derived from foreign or tumour associated antigens, but in the context of MHC class II molecules, which are only expressed by specialized antigen presenting cells (APCs) such as B lymphocytes, macrophages and dendritic cells (DCs).

As a consequence of TCR stimulation of naive CTLs and HTLs by peptide/MHC complexes on APCs, the CTLs mature into effector killer cells capable of lysing (tumour) cells that express the corresponding peptide/MHC class I complex. Activated

HTLs amplify CTL responses by making the APCs more effective at stimulating the naive CTLs and by producing lymphokines that stimulate the maturation and proliferation of CTLs. The potentiating effect of T helper cells occurs both in secondary lymphoid organs where the immune response is initiated and at the tumor site where CTL responses need to be sustained until the tumour cells are eliminated. Thus, one would predict that vaccines should stimulate both tumour-reactive CTLs and HTLs to generate effective antitumour immunity.

Antigens suitable for immunotherapeutic cancer strategies should be highly expressed in cancer tissues and ideally not in normal adult tissues. Expression in tissues that are dispensable for life, however, may be acceptable.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that suitable antigens for immunotherapeutic strategies in the treatment of pancreatic cancer and metastases thereof are provided by the zona pellucida (glyco)proteins. In accordance with the invention, ZP antigens that are able to induce a CD8⁺ and/or CD4⁺ T cell response as well as nucleic acid sequences encoding said antigens, can suitably be used in an immunotherapeutic strategy for therapeutic and/or prophylactic treatment of pancreatic cancer.

The present invention resides in the finding that pancreatic tumour cells display significantexpression of ZP (glyco)proteins, to such extent that these cells are effectively targeted by the immune response elicited by administration of ZP (glyco)protein derived antigens, resulting in decreased growth or even reduced size of primary pancreatic tumours as well as of metastases originating therefrom. The present strategy is equally suitable for preventing metastasis of a pancreatic tumour as well as for preventing the recurrence of pancreatic tumours in previously treated subjects.

Expression of ZP (glyco)protein in pancreatic tumour (derived) cells has never been established before. There is thus no indication in the prior art that pancreatic tumour cells can in fact become the target of a cellular immune response elicited by administering the present ZP (derived) antigens.

The present invention therefore provides for the first time amethods of treating pancreatic tumors in a human comprising immunizing said human with a source of a polypeptide comprising a class I MHC- or class II MHC- restricted native zona pellucida T cell epitope or immunologically active variants thereof, as well as to compositions suitable for use in such methods.

The present invention will be described in more detail hereafter. DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a method for treatment of pancreatic cancer and metastases thereof in a subject by inducing a primary immune response to ZP (glyco)proteins, the method comprising the step of administering to said human a source of a polypeptide, said polypeptide comprising a class I MHC- and/or class II MHC- restricted native zona pellucida T cell epitope that is capable of eliciting a T-cell mediated immune response in vivo or an immunologically active variant thereof. In a particularly preferred embodiment of the invention, the present method is a method for therapeutic treatment.

The naming of the ZP glycoprotein components has been rather inconsistent over the years, employing several criteria, including apparent molecular weight, protein sequence length and sequence identity comparison, which has resulted in a confused nomenclature. Harris et al. [(1994) DNA seq 96: 829-834] proposed a uniform system of nomenclature in which ZP genes were named in order of length of their encoded protein sequence from longest to shortest. Since, under those criteria the mouse ZP genes fell in the order ZP2, then ZPl and then ZP3, a new system was introduced wherein ZP2 became ZPA, ZPl, became ZPB and ZP3 became ZPC. More recently Hughes et al [(1999) BBA-Gene Structure and Expression 1447:303-306], amongst others, reported that the true human orthologue of the known mouse ZPl gene is not ZPB, but that there is a distinct human ZPl gene. It is now generally accepted that there are four distinct (human) ZP glycoprotein families ZPl, ZP2, ZP3 and ZPB [cf. Lefievre et al (2004) Hum. Reprod. 19: 1580-1586]. The ZPB glycoprotein according to this nomenclature is now also referred to as ZP4. This nomenclature is for example applied in the Uniprot/SWISSprot, ensEMBL, BLAST (NCBI), SOURCE, SMART, STRING, PSORT2, CD ART, UniGene and SOSUI databases, all implemented in the Bioinformatic Harvester (http://harvester.embl.de).

In accordance with this the terms ZP1, ZP2, ZP3 and ZP4 are employed herein to denote the four ZP glycoprotein families, wherein ZP2, ZP3 and ZP4 correspond to ZPA, ZPC and ZPB respectively according to the nomenclature proposed by Harris et al. More in particular, the terms hZPl , hZP2, hZP3 and hZP4 as used herein refer to the (glyco)proteins having polypeptide backbones comprised by sequence protocols SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, respectively. In a particularly preferred embodiment of the invention, the ZP (glycol)protein is ZP3 (glycol)protein.

The term 'pancreatic cancer', as used herein, refers to both primary pancreas tumours as well as metastases of said primary pancreatic tumours that may have settled anywhere in the body.

Typically, for the purpose of the present invention, the term 'pancreatic cancer' or 'pancreatic tumour' is synonimous with 'pancreatic neoplastic disease' or 'pancreatic neoplasm' or the like. These terms are deemed entirely interchangeable, although it is noted that for diseases of certain tissues other than the pancreas, the terms 'neoplasm' and 'tumour' may be considered not to coincide entirely. In accordance with the present invention the term 'pancreatic cancer' typically does not include pre-neoplastic conditions, such as hyperplasia, metaplasia, dysplasia or the like.

An important aspect of the present invention is the finding of expression of ZP (glyco)protein on the pancreatic (tumour) cell which allows for an immune response to be elicited against said cells. Neverthelss, as different tumours may have different or altered paterns of gene expression, certain pancreatic tumours not expressing ZP (glyco)proteins to any significant extent might occur as well, as will be understood by the skilled person. Hence, typically, the invention concerns treatment of pancreatic cancer or metastases thereof, expressing ZP (glyco)proteins, preferably ZP3.

However, without wishing to be bound by theory, it is hypothesized that the the phenomenon of epitope spreading or collateral immunization may also be relevant in the methods of the present invention. Colletaral immunization or epitope spreading occurs when, subsequent to induction of a T-cell response against cells expressing ZP glycoproteins, cross-presentation to CD8+ and/or CD4+ T-cells of debris of said ZP- expressing cells, which contain secondary epitopes, by APCs evokes cytotoxic/cytolytic T-cell immune responses against epitopes derived from different antigens, i.e. antigens that were not used for vaccination. Tumor cells expressing these antigens may be attacked in this 'secondary' immune response.

The method according to the invention may constitute the primary treatment or be applied as adjunctive therapy during or following treatment of patients using any of the conventional methods, including for example surgery and chemotherapy and combinations thereof. It is however common knowledge that, often, chemotherapy can be highly immunosuppressive. It will thus be clear to the skilled person that the efficacy of the present method may be lower when following such treatments.

The invention provides methods which are suitably employed for treatment of primary pancreatic cancer and metastases thereof, which is considered herein to constitute 'therapeutic treatment' or 'curative treatment', as well as for preventing metastases and/or recurrence of pancreatic cancer optionally after or in combination with other methods of treatment, such as described before, which is referred to herein as 'prophylactic treatment'. For the methods of the invention, the subject to be treated is preferably a human.

In accordance with the present invention, the ' source of a polypeptide' that is administered to the human according to the present method, may be or comprise a protein or glycoprotein, a digest of the protein or glycoprotein and/or fragments thereof, which may be in a purified form or may be comprised within a crude composition, preferably of biological origin, such as lysates, sonicates or fixates of prokaryotic or eukaryotic cell lines. Alternatively, said source of a polypeptide may be or comprise chemically synthesized (poly)peptides or (poly)peptides that have been produced enzymatically in vitro, which may be in a purified form or may be comprised within a crude composition. The source of the polypeptide may also be or comprise a nucleic acid encoding the polypeptide, from an RNA or DNA template. The RNA or DNA molecules may be 'naked' DNA, preferably comprised in vesicles or liposomes, or may be comprised in a vector. The vector may be any (recombinant) DNA or RNA vector known in the art, and preferably is a plasmid wherein genes encoding latency antigens are operably linked to regulatory sequences conferring expression and translation of the encoded messengers. The vector may also be any DNA or RNA virus, such as but not limited to Adenovirus, Adeno-Associated Virus (AAV), a retrovirus, a lentivirus, modified Vaccinia Ankara virus (MVA) or Fowl Pox virus, or any other viral vector capable of conferring expression of polypeptides comprising latency epitopes to a host. DNA vectors may be non-integrating, such as episomally replicating vectors or may be vectors integrating in the host genome by random integration or by homologous recombination. An example of the construction of plasmids incorporating human ZP2 cDNA, which plasmids could suitably be used in accordance with the present invention can be found in a publication by Martinez et al. [(1996) Journal of Reproduction and Fertility Supplement 50:35-41], which is incorporated herein by reference.

DNA molecules comprising genes encoding the polypeptides according to the current invention, optionally embedded in vectors such as viruses or plasmids, may be integrated in a genome of a host. In a preferred embodiment of the invention, such a host may be a micro-organism. Preferably such a recombinant micro-organism is a Mycobacterium, for instance of the species M. tuberculosis or M. bovis and most preferably M. bovis Bacillus Calmette Guerin (BCG), capable of delivering to a host the polypeptides or fragments thereof according to the invention. Recombinant BCG and methods for recombination are known in the art, for instance in WO2004094469. Such a recombinant micro-organism may be formulated as a live recombinant and/or live attenuated vaccine, as for instance in Jacobs et al. 1987, Nature, 327(6122):532-5). The vector may also be comprised in a host of bacterial origin, such as but not limited to live-attenuated and/or recombinant Shigella or Salmonella bacteria.

The term "epitope" as used herein refers to a portion of an antigen, typically defined by a peptide, which is capable of eliciting a cellular or humoral immune response when presented in a physiologically relevant context in vivo. A "T cell epitope" refers to a peptide or portion thereof that binds to an MHC molecule and is recognized by T cells when presented in MHC molecules. A T cell epitope is capable of inducing a cell mediated immune response via direct or indirect presentation in heterodimeric membrane MHC molecules. Briefly, MHC molecules preferentially bind particular amino acid residues known as "anchor" residues (K. Falk et al., Nature 351 :290-96 (1991)). This characterization permits class I and II MHC recognition epitopes to be identified within any known peptide sequence. In the present context, the term "MHC restricted epitope" is synonymous with T cell epitope. The term "class I MHC restricted epitope", as used herein, refers to peptide sequences recognized by cytotoxic T lymphocytes (also called CD8⁺ cells or CTLs) in association with class I MHC. The term "class II MHC restricted epitope", as used herein, refers to a peptide recognized by helper T cells (also called CD4⁺ cells or HTLs). A "B cell epitope" is a portion of an antigen, typically a peptide, capable of binding to an antigen binding site of an immunoglobulin and therefore capable of stimulating a humoral response without presentation in an MHC molecule. As explained herein before the polypeptide useful in the present invention, or the nucleic acid encoding said polypeptide, comprises at least one T cell epitope. The use of polypeptides that also comprise a B cell epitope is however not excluded from the present invention. The present immunogenic polypeptides may also include multiple T cell epitopes and, optionally a B cell epitope. When multiple epitopes are present in a peptide, the epitopes may be oriented in tandem or in a nested or overlapping configuration wherein at least one amino acid residue may be shared by two or more epitopes.

The polypeptide of the invention preferably includes one or more MHC class I binding epitopes. As is generally known by the skilled person, an antigen comprising a single peptide epitope will be useful only for treating a (small) subset of patients who express the MHC allele product that is capable of binding that specific peptide. It has been calculated that, in humans, vaccines containing CTL epitopes restricted by HLA- Al, -A2, -A3, -A24 and -B7 would offer coverage to approximately 80 % of individuals of most ethnic backgrounds. Therefore, if the present method is used to treat a human , it is particularly preferred that the present source of a polypeptide comprises an effective amount of one or more different polypeptides comprising one, more preferably two, most preferably three MHC class I binding native ZP epitopes selected from HLA-Al, HLA-A2, HLA-A3, HLA-A24 and HLA-B7 restricted epitopes; or homologues thereof or one or more nucleic acid sequence encoding said one or more polypeptides or homologues thereof.

According to another embodiment the polypeptide of the invention preferably includes one or more MHC class II binding epitopes. The most frequently found MHC class II allele products in humans include HLA-DR1, -DR3, -DR4 and -DR7. Accordingly, it is preferred that the present source of a polypeptide, comprises an effective amount of one or more different polypeptides, said one or more different polypeptides comprising one, more preferably two and most preferably three MHC class II binding native ZP epitopes selected from HLA-DR1, HLA-DR3, HLA-DR4 and HLA-DR7 restricted epitopes; or homologues thereof or one or more nucleic acid sequence encoding said one or more polypeptides or homologues thereof.

In still another embodiment, the present source of a polypeptide comprises an effective amount of one or more polypeptides, said one or more polypeptides comprising one or more MHC class I binding epitopes and one or more MCH class II binding epitopes, as described here above; homologues thereof or one or more nucleic acid sequence encoding said polypeptides or homologues thereof. Even, more preferably said source comprises an effective amount of one or more different polypeptides that together include essentially all of the MHC class I and MHC class II binding epitopes comprised in one of the native ZP glycoproteins; or homologues of said one or more polypeptides or one or more nucleic acid sequence encoding said polypeptides or homologues thereof.

In one embodiment, the present source of a polypeptide comprises an effective amount of one or more different immunogenic polypeptides, which one or more different polypeptides together comprise at least 50 %, more preferably at least 70 %, still more preferably at least 80 %, still more preferably at least 90 % and most preferably at least 95 % of the MHC class I and MHC class II restricted binding epitopes comprised in a native ZP glycoprotein; or homologues of said one or more polypeptides or one or more nucleic acid sequences encoding them.

In a preferred embodiment the present source of a polypeptide comprises an effective amount of an immunogenic polypeptide, which polypeptide comprises at least 50 %, more preferably at least 70 %, still more preferably at least 80 %, still more preferably at least 90 % and most preferably at least 95 % of the complete amino acid backbone of a native ZP glycoprotein; or a homologue of said polypeptide or a nucleic acid sequence encoding said polypeptide or homologue thereof

In a particularly preferred embodiment the present source of a polypeptide comprises an effective amount of an immunogenic polypeptide, which polypeptide comprises 90, 95, 97, 98, 99 or 100 % of the complete amino acid backbone of the extracellular domain of a native ZP glycoprotein, preferably of native ZP3 glycoprotein; or a homologue of said polypeptide or a nucleic acid sequence encoding said polypeptide or homologue thereof.

In another particularly preferred embodiment, the source of a polypeptide comprises an effective amount of a plurality of different overlapping polypeptide fragments of a native ZP glycoprotein, which different overlapping polypeptide fragments are between 18-60 amino acids in length and which together comprise at least 50 %, more preferably at least 70 %, still more preferably at least 80 %, still more preferably at least 90 % and most preferably at least 95 % of the complete amino acid backbone of said native ZP glycoproteins; homologues of said polypeptides or one or more nucleic acid sequences encoding said polypeptides or homologues thereof. Typically, the amino acid overlap between the different consecutive 16-80 amino acid polypeptide fragments is at least 7 amino acids, preferably at least 8, more preferably at least 9 and most preferably at least 10 amino acids.

The MHC binding motifs for most common MHC class I and II alleles have been described. These motifs itemize the amino acid residues that serve as MHC binding anchors for specific class I and class II MHC alleles. Sophisticated computer-based algorithms that take into account the MHC binding anchors as well as the amino acids sequence of a peptide are used to predict and quantify the binding affinity of the peptide MHC interaction. Thus, from the input of the known amino acid sequence of Zona Pellucida (glyco)proteins, these algorithms list all potential T-cell epitopes, each with its corresponding predictive binding score. Commonly known bio-informatics tools for these purposes include HLA BIND, SYFPEITHI, NetMHC and TEPITOPE 2000 [see references 1-6]. Alternatively, the skilled artesian will be able to determine HTL and CTL binding epitopes experimentally using standard experimentation (Current Protocols in Immunology, Wiley Interscience 2004).

In some cases it has been observed that the same peptide may bind to several

MHC I or II allele products. In one embodiment, the use of such 'promiscuous' MHC binding peptides in the present method is particularly preferred.

In one embodiment, the current invention provides a method for the induction of a primary immune response to native Zona Pellucida glycoproteins in a human, wherein the method comprises the step of administering to the human a source of a polypeptide, said polypeptide comprising a class I MHC- and/or class II MHC- restricted native zona pellucida T cell epitope or an immunologically active variant thereof, wherein said source of a polypeptide comprises an effective amount of an immunogenic polypeptide selected from Zona Pellucida (glyco)proteins, homologues thereof, and immunologically active fragments of said (glyco)proteins and homologues thereof; or a nucleic acid sequence encoding said immunogenic polypeptide. According to a preferred embodiment said Zona Pellucida (glyco)protein is selected from the group of ZP1 (glyco)protein, ZP2 (glyco)protein, ZP3 (glyco)protein and ZP4 (glyco)protein, more preferably ZP2 (glyco)protein and ZP3 (glyco)protein, most preferably ZP3 (glyco)protein.

According to one particularly preferred embodiment, the source of a polypeptide comprises an effective amount of an immunogenic polypeptide selected from human Zona Pellucida (glyco)proteins, homologues thereof and immunologically active fragments of these (glyco)proteins and their homologues, or a nucleic acid sequence encoding said immunogenic polypeptide. Preferably said human Zona Pellucida (glyco)protein (hZP (glyco)protein) is selected from the group of hZPl (glyco)protein, hZP2 (glyco)protein, hZP3 (glyco)protein and hZP4 (glyco)protein. According to an even more preferred embodiment said (glyco)protein is selected from the group of hZP2 (glyco)protein and hZP3 (glyco)protein, more preferably said (glyco)protein is hZP3 (glyco)protein.

The term 'immunologically active fragments thereof will generally be understood in the art to refer to a fragment of a polypeptide antigen comprising at least an epitope, which means that the fragment at least comprises 4, 5, 6, 7 or 8 contiguous amino acids from the sequence of the polypeptide antigen. According to the present invention the fragment comprises at least a T cell epitope. Thus an 'immunologically active fragment' according to this invention comprises at least 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids from the sequence of the ZP (glyco)protein antigen or homologue or analogue thereof. Still more preferably the fragment comprises both a CTL and a T helper epitope. Most preferably however, the fragment is a peptide that requires processing by an antigen presenting cell, i.e. the fragment has a length of at least about 18 amino acids, which 18 amino acids are not necessarily a contiguous sequence from the polypeptide antigen.

The terms 'homologues thereof, as used herein refer to polypeptides which differ from the naturally occurring polypeptide by minor modifications, but which maintain the basic polypeptide and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes in one or a few amino acids, including deletions (e.g., a truncated version of the peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. As used herein, a homologue or analogue has either enhanced or substantially similar functionality as the naturally occurring polypeptide. Typically, when optimally aligned, such as by the programs GAP or BESTFIT using default parameters, a naturally occurring polypeptide and a homologue thereof share at least a certain percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 8 and gap extension penalty = 2. For proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA. Alternatively percent similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc.

A homologue herein is understood to comprise an immunogenic polypeptide having at least 70 %, preferably at least 80 %, more preferably at least 90 %, still more preferably at least 95 %, still more preferably at least 98 % and most preferably at least 99% amino acid sequence identity with the naturally occurring ZP polypeptides mentioned above and is still capable of eliciting at least the immune response obtainable thereby. A homologue or analogue may herein comprise substitutions, insertions, deletions, additional N- or C- terminal amino acids, and/or additional chemical moieties, such as carbohydrates, to increase stability, solubility and immunogenicity.

According to an embodiment of the invention, the present immunogenic polypeptides as defined herein before, are glycosylated Without wishing to be bound by theory it is hypothesized that by glycosylation of these polypeptides the immunogenicity thereof is increased. Therefore, according to a preferred embodiment, the aforementioned immunogenic polypeptide as defined herein before, is glycosylated, having a carbohydrate content varying from 10-80 wt%, based on the total weight of the glycoprotein or glycosylated polypeptide. More preferably said carbohydrate content ranges from 15-70 wt%, still more preferably from 20-60 wt%. In another embodiment, said glycosylated immunogenic polypeptide comprises a glycosylation pattern that is similar to that of the corresponding zona pellucida glycoprotein (or fragment thereof) of a human. It is hypothesized that this even further increases the immunogenicity of said polypeptide. Thus, in an embodiment it is preferred that the immunogenic polypeptide comprises a glycosylation pattern that is similar to that of the corresponding (fragment of) human ZP glycoprotein. Nevertheless, as is known by the skilled person, recombinant technicques for the production of the immunogenic polypeptide may yield polypeptides which are not glycosylated or which contain different glycosylation patterns, depending on inter alia the choice of the host cells, as will be explained herein below. It will be clear to the skilled person, that the use of recombinant polypeptides, having glycosylation patterns dissimilar to that of the corresponding hZP (fragment), is also entirely within the scope of the present invention and might be preferred in certain embodiments, e.g. for practical reasons.

The present method of immunization preferably comprises the administration of a source of immunogenically active polypeptide fragments, said polypeptide fragments being selected from Zona Pellucida protein fragments and/or homologues thereof as defined herein before, said polypeptide fragments comprising dominant CTL and/or HTL epitopes and which fragments are between 18 and 45 amino acids in length. Peptides having a length between 18 and 45 amino acids have been observed to provide superior immunogenic properties as is described in WO 02/070006. Peptides may advantageously be chemically synthesized and may optionally be (partially) overlapping and/or may also be ligated to other molecules, peptides or proteins. Peptides may also be fused to form synthetic proteins, as in PCT/NL03/00929 and in Welters et al. (Vaccine. 2004 Dec 2;23(3):305-l 1). It may also be advantageous to add to the amino- or carboxy-terminus of the peptide chemical moieties or additional (modified or D-) amino acids in order to increase the stability and/or decrease the biodegradability of the peptide. To improve the immunogenicity / immuno-stimulating moieties may be attached, e.g. by lipidation or glycosylation. To enhance the solubility of the peptide, addition of charged or polar amino acids may be used, in order to enhance solubility and increase stability in vivo.

For immunization purposes the aforementioned immunogenic polypeptides according to the invention may also be fused with proteins such as but not limited to tetanus toxin/toxoid, diphtheria toxin/toxoid or other carrier molecules. The polypeptides according to the invention may also be advantageously fused to heatshock proteins, such as recombinant endogenous (murine) gp96 (GRP94) as a carrier for immunodominant peptides as described in (references: Rapp UK and Kaufmann SH, Int Immunol. 2004 Apr; 16(4):597-605; Zugel U, Infect Immun. 2001 Jun;69(6):4164-7) or fusion proteins with Hsp70 (Triebel et al; W09954464).

The individual amino acid residues of the present immunogenic (poly)peptides of the invention can be incorporated in the peptide by a peptide bond or peptide bond mimetic. A peptide bond mimetic of the invention includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone cross-links. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983). Several peptide backbone modifications are known, these include, ψ [CH₂S], ψ [CH₂ H], ψ [CS H₂ ], ψ [NHCO], ψ [COCH₂ ] and ψ [(E) or (Z) CH=CH]. The nomenclature used above, follows that suggested by Spatola, above. In this context, ψ indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets.

Amino acid mimetics may also be incorporated in the polypeptides. An "amino acid mimetic" as used here is a moiety other than a naturally occurring amino acid that conformationally and functionally serves as a substitute for an amino acid in a polypeptide of the present invention. Such a moiety serves as a substitute for an amino acid residue if it does not interfere with the ability of the peptide to elicit an immune response against the native ZP T cell epitopes. Amino acid mimetics may include nonprotein amino acids, such as β-, γ-, δ-amino acids, β-, γ-, δ—imino acids (such as piperidine-4-carboxylic acid) as well as many derivatives of L-a-amino acids. A number of suitable amino acid mimetics are known to the skilled artisan, they include cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acid and the like. Peptide mimetics suitable for peptides of the present invention are discussed by Morgan and Gainor, (1989) Ann. Repts. Med. Chem. 24:243-252.

In a particularly preferred embodiment, the invention concerns methods and/or uses as defined in the foregoing, wherein the method or use comprises the administration of an immunogenic polypeptide comprising a class I MHC- and/or class II MHC- restricted native zona pellucida 3 (ZP3) T cell epitope capable of eliciting a T- cell mediated immune response, or an immunologically active variant said polypeptide.

In a particularly preferred embodiment, the invention concerns methods and/or uses as defined in the foregoing, wherein the method or use comprises the administration of a polypeptide or glycoprotein corresponding to the extracellular domain of ZP3 glycoprotein, which has an amino acid backbone corresponding to residues 1-383 of ZP3 (SEQ ID NO:3). This extracellular domain fragment is denominated herein as ZP3(l-383). Hence, in a particularly preferred embodiment, the invention concerns methods and/or uses as defined in the foregoing, wherein the method or use comprises the administration of a polypeptide or glycoprotein selected from hZP3(l-383) glycoprotein, a polypeptide having the amino acid backbone of hZP3(l- 383) and homologues and fragments thereof, in accordance with what has been described herein before. Embodiments are also envisaged wherein the method comprises the administration of an expression vector comprising a nucleic acid molecule that encodes the hZP3(l-383) polypeptide or said fragments and/or homologues thereof.

In a particularly embodiment, the invention concerns methods and/or uses as defined in the foregoing, wherein the method or use comprises the administration of an agent selected from hZP3 glycoprotein, recombinant hZP3 glycoprotien, hZP3(l-383) glycoprotein and recombinant hZP3(l-383) glycoprotein. Embodiments are also envisaged wherein the method comprises the administration of an expression vector comprising a nucleic acid molecule that encodes the hZP3 or hZP3(l-383) polypeptide.

In a particularly preferred embodiment, the invention concerns methods and/or uses as defined in the foregoing, wherein the method or use comprises the administration of:

(a) a purified hZP3 glycoprotein, (r)hZP3 glycoprotein, hZP3(l-383) glycoprotein or (r)hZP3(l-383) glycoprotein;

(b) a purified hZP3 polypeptide or hZP3(l-383) polypeptide;

(c) a purified polypeptide or glycoprotein that is an allelic variant of (a) or (b);

(d) an immunologically active fragment of the glycoprotein or polypeptide of (a), (b) or (c), that comprises:

(i) a class I MHC-restricted hZP3 T cell epitope, and/or

(ii) a class II MHC- restricted hZP3 T cell epitope,

which fragment induces an immune response to native hZP3 glycoprotein in a human male; or

(e) a glycoprotein or polypeptide of (a), (b), (c) or (d) that is produced using

recombinant technology; or

(f) an expression vector comprising a nucleic acid molecule that encodes (b), (c) or (d).

In a particularly preferred embodiment, the invention concerns methods and/or uses as defined in the foregoing, wherein the method or use comprises the administration of:

(a) a purified hZP3 glycoprotein;

(b) a purified recombinant hZP3 glycoprotein;

(b) a purified hZP3(l-383) glycoprotein; or

(d) a purified recombinant hZP3(l-383) glycoprotein.

According to a preferred embodiment, the present method comprises the administration of a composition comprising one or more of the present immunogenic polypeptides as defined herein above, and at least one excipient. Excipients are well known in the art of pharmacy and may for instance be found in textbooks such as Remmington's pharmaceutical sciences, Mack Publishing, 1995.

The present method for immunization may further comprise the administration, preferably the co-administration, of at least one adjuvant. Adjuvants may comprise any adjuvant known in the art of vaccination and may be selected using textbooks like Current Protocols in Immunology, Wiley Interscience, 2004.

Adjuvants are herein intended to include any substance or compound that, when used, in combination with an antigen, to immunise a human or an animal, stimulates the immune system, thereby provoking, enhancing or facilitating the immune response against the antigen, preferably without generating a specific immune response to the adjuvant itself. Preferred adjuvants enhance the immune response against a given antigen by at least a factor of 1.5, 2, 2.5, 5, 10 or 20, as compared to the immune response generated against the antigen under the same conditions but in the absence of the adjuvant. Tests for determining the statistical average enhancement of the immune response against a given antigen as produced by an adjuvant in a group of animals or humans over a corresponding control group are available in the art. The adjuvant preferably is capable of enhancing the immune response against at least two different antigens. The adjuvant of the invention will usually be a compound that is foreign to a human, thereby excluding immunostimulatory compounds that are endogenous to humans, such as e.g. interleukins, interferons and other hormones.

A number of adjuvants are well known to one skilled in the art. Suitable adjuvants include e.g. incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-sn-glycero-3- hydroxy-phosphoryloxy)-ethylamine (CGP 19835 A, referred to as MTP-PE), DDA (2 dimethyldioctadecylammonium bromide), polylC, Poly-A-poly-U, RIBI™, GERBU™, Pam3™, Carbopol™, Specol™, Titermax™, tetanus toxoid, diphtheria toxoid, meningococcal outer membrane proteins, diphtheria protein CRM197. Preferred adjuvants comprise a ligand that is recognised by a Toll-like-receptor (TLR) present on antigen presenting cells. Various ligands recognised by TLR's are known in the art and include e.g. lipopeptides (see e.g. WO 04/110486), lipopolysaccharides, peptidoglycans, liopteichoic acids, lipoarabinomannans, lipoproteins (from mycoplasma or spirochetes), double-stranded RNA (poly I:C), unmethylated DNA, flagellin, CpG-containing DNA, and imidazoquinolines, as well derivatives of these ligands having chemical modifications.

The present method for immunization may further comprise the administration, preferably the co-administration, of a CD40 binding molecule in order to enhance a CTL response and thereby enhance the therapeutic effects of the methods and compositions of the invention. The use of CD40 binding molecules is described in WO 99/61065, incorporated herein by reference. The CD40 binding molecule is preferably an antibody or fragment thereof or a CD40 Ligand or a variant thereof, and may be added separately or may be comprised within a composition according to the current invention.For therapeutic applications, the present immunogenic polypetides or nucleic acid sequences encoding them or the present compositions comprising these polypeptides or nucleic acid sequences encoding them are administered to a patient suffering from a pancreatic tumour and possibly metastases thereof or to a patient that has received other methods of treating pancreatic tumours, e.g. any of the conventional methods described herein before, in an amount sufficient to induce a primary autoimmune response directed against native ZP glycoproteins and tissue cells expressing ZP glyoproteins. An amount sufficient to accomplish this is defined as a "therapeutically-" or "prophylactically-effective dose". Such effective dosages will depend on a variety of factors including the condition and general state of health of the patient. Thus dosage regimens can be determined and adjusted by trained medical personnel to provide the optimum therapeutic or prophylactic effect.

In the present method the one or more immunogenic polypeptides are typically administered at a dosage of about 1 μg/kg patient body weight or more at least once. Often dosages are greater than 10 g kg. According to the present invention the dosages preferably range from 1 g kg to 1 mg/kg.

According to one preferred embodiment typical dosage regimens comprise administering a dosage of 1-1000 g/kg, more preferably 10-500 μg/kg, still more preferably 10-150 g/kg, once, twice or three times a week for a period of one, two, three, four or five weeks. According to a preferred embodiment 10-100 μg/kg is administered once a week for a period of one or two weeks. The present method preferably comprises administration of the present immunogenic polypeptides and compositions comprising them via the parenteral or oral route, preferably the parenteral route.

Another embodiment of the invention comprises ex vivo administration of a composition comprising the present immunogenic peptides to mononuclear cells from the patients blood, particularly DC isolated therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin.TM. (Monsanto, St. Louis, Mo.) or GM-CSF IL-4. After pulsing the DC with peptides and washing to remove unbound peptides, the DC are reinfused into the patient. In this embodiment, a composition is provided comprising peptide-pulsed DC which present the pulsed peptide epitopes in HLA molecules on their surfaces. Methods of inducing an immune response employing ex vivo peptide-pulsed DC are well known to the skilled person.

Another aspect of the invention relates to a pharmaceutical preparation comprising as the active ingredient the present source of a polypeptide as defined herein before. More particularly pharmaceutical preparation comprises as the active ingredient one or more of the aforementioned immunogenic polypeptides selected from the group of ZP proteins, homologues thereof and fragments of said ZP proteins and homologues thereof, or, alternatively, a gene therapy vector as defined herein above.

According to a first embodiment a pharmaceutical preparation is provided comprising one or more of the immunogenic polypeptides of the invention. The concentration of said polypeptide in the pharmaceutical composition can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more.

The composition preferably at least comprises a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the immunogenic polypeptides or gene therapy vectors to the patient. For polypeptides, sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions.

According to a particularly preferred embodiment, the present pharmaceutical composition comprises an adjuvant, as defined in more detail herein before. Adjuvants for incorporation in the present composition are preferably selected from the group of ligands that are recognised by a Toll-like-receptor (TLR) present on antigen presenting cells, including lipopeptides (see e.g. WO 04/1 10486), lipopolysaccharides, peptidoglycans, liopteichoic acids, lipoarabinomannans, lipoproteins (from mycoplasma or spirochetes), double- stranded RNA (poly I:C), unmethylated DNA, flagellin, CpG- containing DNA, and imidazoquinolines, as well derivatives of these ligands having chemical modifications. The skilled person will be able to determine the exact amounts of anyone of these adjuvants to be incorporated in the present pharmaceutical preparations in order to render them sufficiently immunogenic. According to another preferred embodiment, the present pharmaceutical preparation may comprise one or more additional ingredients that are used to enhance CTL immunity as explained herein before. According to a particularly preferred embodiment the present pharmaceutical preparation comprises a CD40 binding molecule.

Methods of producing pharmaceutical compositions comprising polypeptides are described in US Patents No 's 5,789,543 and 6,207,718. The preferred form depends on the intended mode of administration and therapeutic application.

For gene therapy, vectors, e.g. a plasmid, phagemid, phage, cosmid, virus, retrovirus, episome or transposable element, comprising a nucleic acid sequence encoding an immunogenic polypeptide as defined herein before may be incorporated into pharmaceutical compositions. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U S Pat. No 5,328,470) or by stereotactic injection (see e.g., Chen et al., PNAS 91 :3054-3057, 1994). The pharmaceutical composition of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The present immunogenic polypeptides are preferably administered parentally. The polypeptides for preparations for parental administration must be sterile. Sterilisation is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilisation and reconstitution. The parental route for administration of the polypeptide is in accordance with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intra-arterial, subcutaneous or intralesional routes. The polypeptide is administered continuously by infusion or by bolus injection. A typical composition for intravenous infusion could be made up to contain 10 to 50 ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a 20% albumin solution and between 10 and 50 mg , preferably between 50 g and 10 mg, of the polypeptide. A typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, 1-10 ml of sterile buffered water and between 10 g and 50 mg, preferably between 50 μg and 10 mg, of the polypeptide of the present invention. Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton, PA, 1980) (incorporated by reference in its entirety for all purposes).

For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that may be added to provide desirable colour, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain colouring and flavouring to increase patient acceptance.

The immunogenic polypeptides for use in the present invention can be prepared using recombinant techniques in which a nucleotide sequence encoding the polypeptide of interest is expressed in suitable host cells such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York; both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J.A., et al. (1985) Gene 34:315 (describing cassette mutagenesis).

An example of the preparation of recombinant human ZPA and ZPB, using baculoviruses can be found in the aforementioned publication by Martinez et al. [(1996) Journal of Reproduction and Fertility Supplement 50:35-41].

Examples of the preparation of recombinant human ZPA and ZPB, using bacteria (E. coli), yeast cells (Pichia pastoris), insect cells (Autographa californica multiple nuclear polyhedrosis virus) and Chinese Hamster ovary cells (CHO) as expression systems are disclosed in a publication by Harris et al. [(1999) Protein Expression and Purification 16:298-307], which is incorporated herein by reference.

An aspect of the invention thus relates to a vector comprising a nucleic acid molecule encoding the present immunogenic polypeptide as defined herein before. Preferably the vector is a replicative vector comprising an origin of replication (or autonomously replication sequence) that ensures multiplication of the vector in a suitable host for the vector. Alternatively the vector is capable of integrating into the host cell's genome, e.g. through homologous recombination or otherwise. A particularly preferred vector is an expression vector wherein a nucleotide sequence encoding a polypeptide as defined above, is operably linked to a promoter capable of directing expression of the coding sequence in a host cell for the vector.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated depending on physiological or developmental conditions. A "tissue specific" promoter is only active in specific types of differentiated cells/tissues.

Expression vectors allow the immunogenic polypeptides as defined above to be prepared using recombinant techniques in which a nucleotide sequence encoding the polypeptide of interest is expressed in suitable cells, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J. A., et al. (1985) Gene 34:315 (describing cassette mutagenesis).

Typically, nucleic acids encoding the desired polypeptides are used in expression vectors. The phrase "expression vector" generally refers to nucleotide sequences that are capable of affecting expression of a gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression can also be used as described herein. DNA encoding a polypeptide is incorporated into DNA constructs capable of introduction into and expression in an in vitro cell culture. Specifically, DNA constructs are suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, e.g., Sf9, yeast, fungi or other eukaryotic cell lines.

DNA constructs prepared for introduction into a particular host typically include a replication system recognised by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. A DNA segment is "operably linked" when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, DNA sequences that are operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading phase. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.

The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001, supra). The transcriptional regulatory sequences typically include a heterologous enhancer or promoter that is recognised by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001, supra). Expression vectors include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and in Metzger et al. (1988) Nature 334: 31-36. For example, suitable expression vectors can be expressed in, yeast, e.g. S.cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. Since prokaryotes do not possess the organelles necessary for glycosylation, polypeptides produced by prokaryotes will not have carbohydrate side chains. Eukaryotes do have the glycosylation machinery, but yeast cells will give a different glycosylation pattern than mammalian cells. It is therefore preferred to use an expression system which gives the most "natural" glycosylation pattern. Towards this end mammalian cells are most preferred. Cell lines having glycosylation machinery similar to that of a human can be particularly useful, since it is hypothesized that antigens according to the present invention having a glcyocylation pattern similar to that of the corresponding human Zona Pellucida glycopolypeptides may have increased immunogenicity. Suitable cell lines include CHO cells, see, e.g., U.S. Pat. No 5,272,070 and in particular human ovary or follicle cell lines, cf. WO 99/42581.

In vitro mutagenesis and expression of mutant proteins are described generally in Ausubel et al. (1987, supra) and in Sambrook and Russell (2001, supra). Also see, Kunkel (1985, supra; describing site directed mutagenesis) and Roberts et al. (1987, supra; describing cassette mutagenesis).

Another method for preparing the present immunogenic polypeptides is to employ an in vitro transcription/translation system. DNA encoding a polypeptide is cloned into an expression vector as described supra. The expression vector is then transcribed and translated in vitro. The translation product can be used directly or first purified. Polypeptides resulting from in vitro translation typically do not contain the post- translation modifications present on polypeptides synthesised in vivo, although due to the inherent presence of microsomes some post-translational modification may occur. Methods for synthesis of polypeptides by in vitro translation are described by, for example, Berger & Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, CA, 1987 (incorporated herein by reference in its entirety).

A further aspect of the invention thus relates to a host comprising a vector as defined above. The host cells may be prokaryotic or eukarotic host cells as indicated above. The host cell may be a host cell that is suitable for culture in liquid or on solid media. Alternatively, the host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal, preferably a non-human animal.

A further aspect the invention relates to a method for producing the present immunogenic polypeptide as defined above. The method comprises the step of culturing a host cell as defined above under conditions conducive to the expression of the polypeptide. Optionally the method may comprise recovery the polypeptide. The polypeptide may e.g. be recovered from the culture medium by standard protein purification techniques, including a variety of chromatography methods known in the art per se.

Another aspect of the invention relates to a transgenic animal comprising in its somatic and germ cells a vector as defined above. The transgenic animal preferably is a non-human animal. Methods for generating transgenic animals are e.g. described in WO 01/57079 and in the references cited therein. Such transgenic animals may be used in a method for producing a polypeptide as defined above, the method comprising the step of recovering a body fluid from a transgenic animal comprising the vector or a female descendant thereof, wherein the body fluid contains the polypeptide, and, optionally recovery of the polypeptide from the body fluid Such methods are also described in WO 01/57079 and in the references cited therein. The body fluid containing the polypeptide preferably is blood or more preferably milk.

Yet another aspect of the invention relates to a transgenic plant comprising in its cells a vector as defined above. Methods for generating transgenic plants are e.g. described in U.S. 6,359, 196 and in the references cited therein. Such transgenic plants may be used in a method for producing a polypeptide as defined above, the method comprising the step of recovering a part of a transgenic plant comprising in its cells the vector or a part of a descendant of such transgenic plant, whereby the plant part contains the polypeptide, and, optionally recovery of the polypeptide from the plant part. Such methods are also described in U.S. 6,359, 196 and in the references cited therein.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The invention is further illustrated in the following examples, which are not intended to limit the scope of the invention in any manner.

EXAMPLES

Example 1

The expression of ZP proteins in human pancreatic tumour tissue is determined using immunohistochemical methods. Samples from pancreatic tumour tissues originating from different patients were obtained from a pathology institute in the Netherlands.

For the immunohistochemical determination, rabbit polyclonal antibodies to human recombinant ZP1, ZP2 and ZP3 proteins are used.

Immature oocytes collected from antral follicles following ovarian stimulation for rVF were stained as positive controls. In addition, a sample of liver tissue was used as a negative control.

The following protocol is used for all samples

Deparaffine/hydrate sections:

a. Indubate sections in three washes of histoclear for 5 minutes each b. Incubate sections in two washes of 100% ethanol for 10 minutes each c. Incubate sections in two washes of 95% ethanol for 10 minutes each d. Incubate sections in two washes of 70% ethanol for 10 minutes each Wash sections twice in dEhO for 5 minutes each

Wash sections in PBS for 5 minutes each

For antigen unmasking, heat sections in 10 mM sodium citrate buffer (pH 6.0) (see over for recipe) for 1 min follow by medium power 9 min. Keep slides fully immersed in buffer and maintain temperature at or just below boiling. Cool slides for 20 min.

Was sections in d hO three times for 5 min each 6. Incubate sections in 1% hydrogen peroxide buffer (see over for recipe) for 30 min

7. Wash sections in dfhO three times for 5 minutes each

8. Wash sections in PBS for 5 minutes

9. Block each section with 100-400 μΐ blocking solution (see over for recipe) for 1 hour at room temperature

10. Remove blocking solution end add 100-400 μΐ diluted primary antibody to each section (dilute antibody (1 :250-1 :500) in diluted (1 in 4) blocking solution). Incubate overnight at +4°C

Day 2

1 1. Remove antibody solution and wash sections in PBS three times for 5 minutes each

12. Add 100-400 μΐ secondary antibody 1 : 1000, diluted in blocking solution (again dilute 1 in 4), to each section. Incubate 1 hour at room temperature

13. If using ABC biotin/avidin method, make ABC reagent according to the

manufactures instructions and incubate solution for 30 minutes at room temperature

14. Remove secondary antibody solution and wash sections in PBS three times for 5 minutes each

15. Add 100-400 μΐ ABC reagent to each section and incubate for 1 hour at room temperature

16. Remove ABC reagent and wash sections in PBS three times for 5 minutes each

17. Add 100-400 μΐ DAB reagent to each section and monitor staining closely.

18. As soon as the section turns brown, immerse slides in dlHhO

19. Counterstain sections in haematoxylin for 10 seconds

20. Wash sections in dfbO two times for 5 minutes each

21. Dehydrate sections:

a. Incubate sections in 70% ethanol two times for 10 seconds each b. Incubate sections in 95% ethanol two times for 10 seconds each c. Repeat in 100% ethanol, incubating sections two times for 10 seconds each

d. Repeat in histoclear, incubating sections two times for 10 seconds each

22. Mount coverslips and leave to dry before visualising. Solutions and reagents

• Xylene (or Hi stocl ear)

· Ethanol

• Distilled H₂0

• Haematoxylin

. 1 OX PBS (Phosphate Buffered Saline):

• 0.58 M sodium phosphate dibasic (Na₂HP04), 0.17 M sodium phosphate monobasic (NaH₂P0₄), 0.68 M NaCl. To prepare 1 liter of 10X PBS: Combine 82.33 g

Na₂HP0₄*4H₂0, 23.45 g NaH₂P0₄*H₂0 and 40 g NaCl. Adjust pH to 7.4.

• 10 mM Sodium Citrate Buffer:

To prepare 1 liter, add 2.94 g sodium citrate to 1 liter dH₂0. Adjust pH to 6.0

• 1 % Hydrogen Peroxide (oxidation) buffer:

In 50ml: 15μ1 Triton-X, 10ml Methanol, 40ml 1% H₂0₂ (final cone.)

• Blocking solution:

10% FBS and 10% BSA in PBS

• ABC reagent (Vectastain ABC kit, Vector laboratories, Inc., Burlingame, CA):

Prepare acoording to manufacturer's instructions 30 minutes before use

· DAB reagent:

Use as per manufacturers instructions

In the positive controls, all three antibodies detect proteins in the ZP surrounding the human oocyte. The three proteins are also present in the oocyte cytoplasm. No positive staining is detected in sections of the pancreatic tumour tissue samples when the primary antibody is omitted. No positive staining is observed, with each of the ZP antibodies, in liver tissue.

In the pancreatic tumour samples, presence of the ZP is confirmed by areas of the tissue staining positive for ZPl, ZP2 and/or ZP3, with intensities varying among the samples obtained from different patients. These tumours staining positive for ZP expression can be treated by immunisation with the respective ZP-antigens in accordance with the present invention.

Example 2 The expression of ZP3 proteins in human pancreatic tumour tissue is determined using immunohistochemical methods. Samples from pancreatic cancer ('PaCa') sampels originating from different patients were obtained from a pathology institute in the Netherlands.

Formalin-fixed paraffin sections of pancreatic tumors (5 μιη) were deparaffinized in changes of xylene, rehydrated in decreasing concentration of ethanol (from absolute, through 96%, 70%, 50% to dH₂0) and boiled in 10 mM citric acid (pH 6.0) for 15 min for antigen retrieval. After cooling down, sections were washed in TBST (TBS with 0.05% Tween 20) 3 times for 5 minutes each. Endogenous peroxidase activity was blocked by incubating tissue sections in methanol with 3% H2O2 for 15 min at room temperature (RT). Sodium borohydride (1 mg/ml, Sigma Aldrich, Munich, Germany) was used to quench free aldehyde groups (15 min). To reduce non-specific background staining tissue sections were incubated with blocking solutions 3% BSA in TBST for 1 h at RT. Thereafter, sections were incubated with 5μg/ml mouse monoclonal anti-hZP3 antibody in blocking buffer overnight at 4°C. The next day sections were washed in TBST 3 times for 5 minutes each and incubated with EnVision anti-mouse polymer (Dako, Glostrup, Denmark) for 30 min at RT After washing sections in TBST 3 x 5 min immunoreaction was visualized by incubating sections with 3 '3-diaminobenzidine tetrahydrochloride (DAB, Dako) for 5 min. Hematoxylin was used as counterstain. After the staining sections were dehydrated and mounted with Pertex (Histolab Products AB, Gothenburg, Sweden).

The results of the staining are shown in figures 1-10. As can be seen in these figures, the ZP3 monoclonal Ab gave very specific cytoplasmic staining of the Carcinoma cells. Pancreatic cancer (PaCa) samples stained ZP3 positive (n = 7/1 1).

Description of the Figures

Fig 1 : PaCa cells lOx magnification

Fig 2: same slide as Fig. 1 with 40X magnification, cytoplasmic staining of the PaCa carcinoma cells.

Fig 3 : PaCa cancer cells (arrow heads), 10X and besides necrotic tissue/blood clusters Fig. 4: same slide as Fig. 3 with 40x magnification

Fig. 5 : Pancreatic intraepithelial neoplasia grade 3 (PanIN3) cells stained with ZP3, one can see that the ZP3 stained cells are trying to infliltrate, and after the infiltration they will become carcinoma cells Fig 6: Direct smear of ductal adrenocarcinoma. arrowheads showing the carcinoma cells stained with ZP3

Fig 7: A positive control - human ovary, oocytes stained with ZP3

Fig. 8: Higher magnification human ovary +ve control ZP3 layer of the oocyte stained with ZP3 monoclonal Ab

Fig 9: lower magnification

Fig 10: Higher magnification of Human Spleen stained with ZP3 as negative control REFERENCES

1. Parker, K. C, M. A. Bednarek, and J. E. Coligan. 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152: 163. HLA BIND

(http://bimas.cit.nih.gov/molbio/hla_bind/)

2. Rammensee, Friede, Stevanovic, MHC ligands and peptide motifs: 1 st listing, Immunogenetics 41, 178-228, 1995; SYFPEITHI (http://www.syfpeithi.de/) and

Rammensee, Bachmann, Stevanovic MHC ligands and peptide motifs. Landes Bioscience 1997 (International distributor - except North America: Springer Verlag GmbH & Co. KG, Tiergartenstr. 17, D-69121 Heidelberg

3. Buus S, Lauemoller SL, Worning P, Kesmir C, Frimurer T, Corbet S, Fomsgaard A, Hilden J,Holm A, Brunak S. Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee' artificial neural network approach, in Tissue Antigens., 62:378-84, 2003; NetMHC (http://www.cbs.dtu.dk/services/NetMHC/)

4. Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, Brunak S, Lund O , Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci., 12: 1007-17, 2003.

5. Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach, Nielsen M, Lundegaard C, Worning P, Hvid CS, Lamberth K, Buus

5, Brunak S, Lund O., Bioinformatics, 20(9): 1388-97, 2004.

6. Sturniolo, T. et al., Nature Biotechnology 17, 555-562, 1999, Generation of tissue-specific and promiscuous HLA ligand databases using DNA chips and virtual

HLA class II matrices; TEPITOPE (http://www.vaccinome.com/pages/597444/).

Claims

Use of a source of a polypeptide comprising a class I MHC- and/or class II MHC- restricted native zona pellucida T cell epitope or immunologically active variant thereof for the manufacture of a medicament for use in a method for treatment of pancreatic cancer and/or metastases thereof in a subject.

Use according to claim 1, wherein the method of treatment is a method of therapeutic treatment.

Use according to claim 1 or 2, wherein said source of a polypeptide comprises an effective amount of an immunogenic polypeptide selected from hZP3 (glyco)protein, homologues thereof and immunologically active fragments of said hZP3 (glyco)proteins and homologues thereof; or a nucleic acid sequence encoding said immunogenic polypeptide.

Use according to claim 1 or 2, wherein said source of a polypeptide comprises an effective amount of an immunogenic polypeptide selected from hZP3(l-383) (glyco)protein, homologues thereof and immunologically active fragments of said hZP3(l-383) (glyco)proteins and homologues thereof; or a nucleic acid sequence encoding said immunogenic polypeptide..

Use according to claim 1 or 2, wherein said immunogenic polypeptide is selected from hZP3 glycoprotein, recombinant hZP3 glycoprotein, hZP3(l-383) glycoprotein and recombinant hZP3(l-383) glycoprotein..

Use according to any one of claims 1-5, wherein said source of a polypeptide is selected from the group consisting of:

(a) purified hZP3 glycoprotein and hZP3(l-383) glycoprotein;

(b) purified hZP3 polypeptide and hZP3(l-383) polypeptide;

(c) purified polypeptides and glycoproteins that are allelic variants of (a) or

(b);

(d) immunologically active fragments of the glycoproteins and polypeptides of (a), (b) or (c), that comprises: (i) a class I MHC-restricted hZP3 T cell epitope, and/or

(ii) a class II MHC- restricted hZP3 T cell epitope,

which fragment induces an immune response to native hZP3 glycoprotein in a human male;

(e) glycoproteins and polypeptides as defined in (a), (b), (c) or (d) that are produced using recombinant technology; and

(f) expression vectors comprising a nucleic acid molecule that encodes a polypeptide as defined in (b), (c) or (d).

7. Use according to claim 1 or 2, wherein said source of a polypeptide comprises an effective amount of one or more different immunogenic polypeptides, said one or more immunogenic polypeptides together comprising at least 50 %, more preferably at least 70 %, of the MHC class I and MHC class II restricted binding epitopes comprised in native ZP3 glycoprotein; homologues of said one or more immunogenic polypeptides; or one or more nucleic acid sequences encoding said polypeptides or homologues thereof.

8. Use according to claim 1 or 2, wherein said source of a polypeptide comprises an effective amount of an immunogenic polypeptide comprising at least 50 %, more preferably at least 70 % of the complete amino acid backbone of a native ZP3 glycoprotein; a homologue of said immunogenic polypeptide; or a nucleic acid sequence encoding said polypeptide or homologue thereof.

9. Use according to claim 1 or 2, wherein said source of a polypeptide comprises an effective amount of a plurality of different overlapping polypeptide fragments of a native ZP3 glycoprotein, which different overlapping polypeptide fragments are between 18 and 60 amino acids in length and which together comprise at least 50 %, more preferably at least 70 %, of the complete amino acid backbone of said native ZP3 glycoprotein; homologues of said polypeptide fragments; or one or more nucleic acid sequence encoding said polypeptide fragments or homologues thereof.

10. Use according to anyone of the preceding claims wherein the polypeptide is glycosylated.

1 1. Use according to anyone of the preceding claims, wherein the method comprises the administration, preferably the co-administration, of at least one adjuvant.

12. Use according to anyone of the preceding claims, wherein the subject is suffering from pancreatic cancer or metastases thereof, expressing ZP3 glycoprotein.

13. Pharmaceutical composition comprising a source of a polypeptide as defined in any one of claims 1-9 for use in a method for treatment of pancreatic cancer and/or metastases thereof in a subject.

14. Pharmaceutical composition according to claim 13 comprising an adjuvant.

15. Pharmaceutical composition according to claim 13 or 14, wherein the immunogenic polypeptide is hZP3, a homologue thereof or an immunologically active fragment of said protein or homologue thereof.