WO2008040759A1

WO2008040759A1 - Method for down-regulation of cripto

Info

Publication number: WO2008040759A1
Application number: PCT/EP2007/060502
Authority: WO
Inventors: Valéry RENARD; Dana Leach; Steen Klysner; Peter Birk Rasmussen; Finn Stausholm Nielsen; Tomas Bratt; Bjørn VOLDBORG; Florence Dal Degan
Original assignee: Pharmexa A/S
Priority date: 2006-10-03
Filing date: 2007-10-03
Publication date: 2008-04-10

Abstract

The present invention provides for immunogenic variants of CRIPTO which are useful in active specific immunotherapy against diseases that are characterized by overexpression of CRIPTO. The invention also relates to methods of treating such diseases (for instance cancer) as well as to various tools in molecular biology that assist in the provision of the immunogenic variants.

Description

METHOD FOR DOWN-REGULATION OF CRIPTO

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic immunotherapy, and in particular to the field of active immunotherapy targeted at down-regulating the autologous ("self") protein CRIPTO. The invention thus provides novel and improved immunogenic variants of the CRIPTO protein as well as the necessary tools for the preparation of such variants. The invention further relates to methods of immunotherapy and anti-cancer therapy as well as compositions useful in such methods.

BACKGROUND OF THE INVENTION

CRIPTO is a naturally occurring cell-surface protein that is associated with signal transduction pathways and is up-regulated in many human cancers, where it is associated with the maintenance of the transformed state of tumor cells.

Indeed, tumors of different origins were shown to over-express CRIPTO, while CRIPTO seems absent from normal tissues. In vitro findings suggested that CRIPTO over expression by cancer cells might promote tumor growth.

The therapeutic targeting of CRIPTO with monoclonal antibodies (mAbs) has been shown to inhibit the in vivo growth of human tumors transplanted into immunodeficient mice.

CRIPTO is a member of the EGF-CFC family of proteins that includes human CRIPTO and Criptic, murine CRIPTO and Criptic, frog FRL-I, zebrafish one-eyed pinhead protein and chick CRIPTO. These proteins are characterized by two extracellular cysteine-rich structural motifs: an epidermal growth factor (EGF)-like domain, and a CRIPTO/FRL-1/Cryptic (CFC) domain, the latter of which is considered unique to this family. CRIPTO is expressed at the cell surface and attached to the cell membrane through a glycosyl-phosphatidylinositol (GPI) linkage.

Genetic studies in mice and fish have revealed that EGF-CFC proteins play essential roles in early embryonic development in specification of the anterior-posterior and left-right body axes, as well as in the formation of the primary germ layers during gastrulation. Further studies have demonstrated that EGF-CFC proteins act as coreceptors for Nodal, a member of the transforming growth factor beta (TGF-beta superfamily). Nodal signals through the type I activin receptor ActRIB (ALK4) and the type II receptors ActRIIA and ActRIIB. Membrane-bound CRIPTO appears to recruit Nodal to an activin receptor complex composed of a dimer of ActRIB and a dimeric type II activin receptor. The interaction of CRIPTO with ActRIB requires its CFC motif, whereas CRIPTO binding to Nodal utilizes the EGF-like domain and requires post-translational modification by O-fucosylation found on certain EGF motif-containing proteins.

In the adult, only very low levels of CRIPTO mRNA are detected by RT-PCR in different organs (spleen, testis, heart, lung, brain, and mammary gland) but no function has been described in any normal tissue, except for the mammary gland. Different levels of CRIPTO expression are found in the virgin, pregnant, lactating, involuting and aging mammary gland. As assessed by western blot analysis, immunocytochemistry and in situ hybridization, CRIPTO expression is enhanced by three- to five-fold during pregnancy and lactation and is totally lost during involution. CRIPTO is also upregulated in ductal epithelial cells in the aging mammary gland of Balb/c mice that exhibit spontaneous mammary tumor development.

Expression/overexpression of CRIPTO in cancer tissues of epithelial origin has been reported since 1991. The reports concern breast, pancreatic, colon, lung, and ovarian cancers^1'5. Immunostaining performed on cancer tissues showed that CRIPTO was highly expressed in 50-80% of the tested tissues. An 11-fold increase in CRIPTO mRNA levels was also reported in pancreatic cancer tissues as compared to normal tissues 2. However, CRIPTO overexpression does not seem to be associated with tumor stage or overall survival and has not been described as a prognostic marker.

The potential significance of CRIPTO overexpression in cancer was first investigated in mammary epithelial and tumor cell lines transfected with CRIPTO. These experiments showed that CRIPTO overexpression increased the in vitro cell proliferation rate of transformed and non-transformed cells, it lead to increased cell density, and eventually to transformation, while it did not induce in vivo tumorigenesis^{5 10}.

Signaling pathways activated by CRIPTO and responsible for cellular proliferation and transformation still needs clarification. Recently, it was shown that CRIPTO forms complex with activin and type II activin receptors, which seems to prevent the formation of activin/ activin receptors complexes¹². In addition, transfection of the CRIPTO gene into different cell lines expressing activin receptors resulted in the inhibition of activin signaling. As activin is a potent inhibitor of cell growth in multiple cell types, these data provide a mechanism that may partially explain the oncogenic action of CRIPTO. In addition to Nodal and activin receptors, CRIPTO was shown to interact with another GPI- linked surface molecule, Glypican-1, and to activate the cytoplasmic tyrosine kinase c-Src that has been implicated in cancer development. Expression of Glypican-1 is elevated in human breast cancer, whereas its expression is low in normal breast tissue¹³. Because CRIPTO is also overexpressed in breast cancer, Glypican may act to promote the growth- promoting effect of CRIPTO in breast cancer cells.

Experimental therapeutic targeting of CRIPTO suggests that the molecule plays an important role in cancer biology. Inhibition of CRIPTO protein expression by antisense oligonucleotides showed anti-proliferative effect on tumor cells, both in vitro and in vivo (Xenograft models), as well as additive effect with the anti-EGFR monoclonal antibody Erbitux in one cancer model^{14 15}.

In addition, the anti-tumor activity of monoclonal antibodies (mAbs) specific for CRIPTO in animal models of cancer was recently reported¹⁷' ¹⁸. IgM mAbs raised against a 17-mer CRIPTO peptide, mapping to the EGF-like domain, inhibited in vitro and in vivo growth of different tumor cell lines expressing CRIPTO¹⁷. Yet different mAbs raised against a recombinant fusion protein (IgGl)Fc-CRIPTO, showed similar anti-tumor activity in different animal models. Curiously in these last models, mAbs specific for the CFC domain of CRIPTO showed greater anti-tumor activity than mAbs specific for the EGF-like domain. Taken together, the above data suggest that the targeting of the two individual domains may result in a therapeutic effect, depending on the tumor model/context, and also suggest that a vaccine approach inducing polyclonal antibodies (potentially targeting both the EGF-like and the CFC domains of CRIPTO) may show greater efficacy than mAbs.

The cloned wild type (wt) human CRIPTO sequence (SEQ ID No 16) has 188 amino-acid residues and a calculated molecular weight of 36 kDa with fucosylations and glycosylates and 21 kDa without glycosylations¹⁹.

The three-dimensional crystal structure of CRIPTO has not been published. From sequence alignments of cloned CRIPTO proteins, six individual protein elements are identified. N- terminally, the CRIPTO sequence starts with a signal peptide targeting the endoplasmic reticulum; Metl-Ala29. This is followed by a long domain with unknown function; Gly30- Pro69. Then a short sequence, Met70-Thr81, is present next to the EGF-like domain in Cys82-Arglll. The CFC domain is found at Cysll5-Aspl50. In between the EGF and CFC domains, a three residues linker region is present, Lysll2-Asnll4. A GPI link for membrane attachment is located in the C-terminal of the CRIPTO protein; Glyl51- Tyrl88. CRIPTO amino-acid sequence shows a glycosylation site at Asn79 and a functionally important fucosylation site at Thr88.

As the crystal structure of CRIPTO is unknown, researchers have made a model of CRIPTO, based on published NMR coordinates of the EGF and the CFC domains²⁰. This model covers barely 50% of the total structure. The model shows several loop regions held together by disulfide bonds (Figure 1). This fits well with the secondary structure prediction analysis we have performed.

It is well known that each of the EGF-like and CFC domains have a much conserved disulfide bond pattern with three disulfide bridges in each domain. By peptide-mapping it has been shown that these six disulfide bonds are located in human CRIPTO at positions: Cys82- Cys89; Cys83-Cys95; Cys97-CyslO6; Cysll5-Cysl33; Cysl28-Cysl49 and Cysl31- Cysl40²⁰. Furthermore, there are two free cystein residues in the terminal ends: Cys3 and Cyslβl. The degree of standard structure elements like α-helixes and β-sheets in CRIPTO is limited. The molecule is held together by the six disulfide-bridges. Some structure transformation could occur after binding of CRIPTO to its ligand(s).

Use of active immunotherapy ("vaccination") as a means of curing or alleviating disease has received growing attention over the last 2 decades. Notably, the use of active immunotherapy as a means for breaking tolerance to autologous proteins that are somehow related to a pathological (or otherwise undesired) physiologic condition has been known since the late seventies where the first experiments with antifertility vaccines where reported.

Vaccines against autologous antigens have traditionally been prepared by "immunogenizing" the relevant self-protein, e.g. by chemical coupling ("conjugation") to a large foreign and immunogenic carrier protein (cf. US 4,161,519) or by preparation of fusion constructs between the autologous protein and the foreign carrier protein (cf. WO 86/07383). In such constructs, the carrier part of the immunogenic molecule is responsible for the provision of epitopes for T-helper lymphocytes ("T_H epitopes") that render possible the breaking of autotolerance.

Later research has proven that although such strategies may indeed provide for the breaking of tolerance against autologous proteins, a number of problems are encountered. Most important is the fact that the immune response that is induced over time will be dominated by the antibodies directed against the carrier portion of the immunogen whereas the reactivity against the autologous protein often declines, an effect that is particularly pronounced when the carrier has previously served as an immunogen - this phenomenon is known as carrier suppression (cf. e.g. Kaliyaperumal et al. 1995., Eur. J. Immunol 25, 3375- 3380). However, when using therapeutic vaccination it is usually necessary to re-immunize several times per year and to maintain this treatment for a number of years and this also results in a situation where the immune response against the carrier portion will be increasingly dominant on the expense of the immune response against the autologous molecule.

Further problems involved when using hapten-carrier technology for breaking autotolerance is the negative steric effects exerted by carrier on the autologous protein part in such con- structs: The number of accessible B-cell epitopes that resemble the conformational patterns seen in the native autologous protein is often reduced due to simple shielding or masking of epitopes or due to conformational changes induced in the self-part of the immunogen. Finally, it is very often difficult to characterize a hapten-carrier molecule in sufficient detail.

WO 95/05849 provided for a refinement of the above-mentioned hapten-carrier strategies. It was demonstrated that self-proteins wherein is in-substituted as little as one single foreign T_H epitope are capable of breaking tolerance towards the autologous protein. Focus was put on the preservation of tertiary structure of the autologous protein in order to ensure that a maximum number of autologous B-cell epitopes would be preserved in the immunogen in spite of the introduction of the foreign T_H element. This strategy has generally proven extremely successful inasmuch as the antibodies induced are broad-spectre as well as of high affinity and that the immune response has an earlier onset and a higher titre than that seen when immunizing with a traditional carrier construct.

WO 00/20027 provided for an expansion of the above principle. It was found that introduction of single T_H epitopes in the coding sequence for self-proteins could induce cytotoxic T-lymphocytes (CTLs) that react specifically with cells expressing the self-protein. The technology of WO 00/20027 also provided for combined therapy, where both antibodies and CTLs are induced - in these embodiments, the immunogens would still be required to preserve a substantial fraction of B-cell epitopes.

OBJECT OF THE INVENTION

It is an object of the invention to provide for immunogenic analogues of CRIPTO as well as to provide for methods for inducing immunity against this protein, notably in the treatment of cancer such as solid tumours. Finally, it is also objects of the invention to provide for means and measures that are useful when preparing or utilising the immunogens.

SUMMARY OF THE INVENTION

The CRIPTO system provides an attractive target for therapeutic intervention. The anti-tumor activity of monoclonal antibodies (mAbs) specific for CRIPTO in animal models of cancer was recently reported¹⁷' ¹⁸

The present inventors have devised an attractive alternative based on the vaccine principle, i.e. to harness the patient's own immune system to produce antibodies to neutralize CRIPTO via a vaccine approach that bypasses immunological tolerance and can be used to generate neutralizing antibodies to self-proteins like CRIPTO. This is achieved by active immunization with recombinant CRIPTO proteins modified to contain a highly immunodominant and promiscuous foreign peptide recognized by T helper cells. Due to functional tolerance, only T helper cells that recognize the inserted foreign epitope become activated. These activated T helper cells can then provide the necessary signals for CRIPTO-specific B cells to differentiate into antibody-secreting plasma cells. The antibodies produced by these plasma cells are then capable of neutralizing or clearing CRIPTO in vivo. In general terms, this process is inherently similar to any normal immune response driven by T cells responding to foreign antigens. The present approach simply harnesses these foreign-specific T helper cells to drive the anti- CRIPTO immune response. Importantly, in the absence of this foreign T helper response the anti-CRIPTO immune response wanes.

In its most general scope, the invention relates to a method for in vivo down-regulation of CRIPTO activity in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of

at least one autologous CRIPTO protein or an autologous CRIPTO polypeptide or subsequence thereof which has been formulated so that immunization of the animal with the CRIPTO protein or CRIPTO polypeptide or subsequence thereof induces production of antibodies against the animal's autologous CRIPTO protein, and/or

at least one CRIPTO analogue, which comprises a CRIPTO polypeptide wherein is introduced at least one modification in the CRIPTO amino acid sequence which has as a result that immunization of the animal with the analogue induces production of antibodies against the animal's autologous CRIPTO protein.

The invention further provides for nucleic acid fragments (such as DNA fragments) encoding such immunogenic analogues and also to vectors including such DNA fragments.

The invention also provides for transformed cells useful for preparing the analogues.

The invention further provides for immunogenic compositions comprising the analogous or the vectors of the invention.

Also provided by the invention are methods of treatment, where CRIPTO is down-regulated and to treatment of specific diseases, such as malignant neoplasms.

The invention also provides a method for the preparation of a CRIPTO vaccine capable of inducing in vivo down regulation of CRIPTO activity in an animal, including a human being, said method comprising either

a) Introducing at least one modification into at least one template molecule, wherein said template molecule is selected from the group comprising an autologous CRIPTO protein, a CRIPTO protein, CRIPTO polypeptide and a CRIPTO analogue,

and/or

b) combining at least one autologous CRIPTO protein, an autologous CRIPTO polypeptide or subsequence thereof, a CRIPTO analogue, or the modified template prepared in a), with at least one agent which effects targeting and/or presentation of the autologous CRIPTO protein or an autologous CRIPTO polypeptide to an antigen presenting cell

(APC) or a B-lymphocyte and/or stimulates the immune system.

The invention also provides for a CRIPTO vaccine, such as CRIPTO analogues suitable for use as a CRIPTO vaccine, prepared according to the above method.

Therefore the invention also provides for a CRIPTO analogue which is derived from an animal CRIPTO polypeptide wherein is introduced a modification which has as a result that immunization of the animal with the analogue induces production of antibodies against the CRIPTO polypeptide. Such a CRIPTO analogue is considered a CRIPTO vaccine or suitable for use as a CRIPTO vaccine.

The invention also provides for an immunogenic composition (such as a CRIPTO vaccine) comprising an immunogenically effective amount of a CRIPTO polypeptide autologous in an animal, said CRIPTO polypeptide being formulated together with an immunologically acceptable adjuvant so as to break the animal's autotolerance towards the CRIPTO polypeptide, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle.

The invention also provides for an immunogenic composition (such as a CRIPTO vaccine) comprising an immunogenically effective amount of a CRIPTO analogue, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant.

The invention provides for the following polypeptides: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15.

The invention also provides for nucleic acid fragments which encode CRIPTO analogues and CRIPTO protein vaccines, and vectors which comprise said nucleic acid fragments.

The invention also provides for host cells which comprise nucleic acid fragments which encode CRIPTO analogues and CRIPTO protein vaccines, such as vectors comprising said nucleic acid fragments.

The invention also provides for nucleic acid fragments which encode for the following polypeptides: SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

In the following, a number of terms used in the present specification and claims will be defined and explained in detail in order to clarify the metes and bounds of the invention. The terms "T-lymphocyte" and "T-cell" will be used interchangeably for lymphocytes of thymic origin that are responsible for various cell mediated immune responses as well as for helper activity in the humeral immune response. Likewise, the terms "B-lymphocyte" and "B- cell" will be used interchangeably for antibody-producing lymphocytes.

"An immunogenic analogue" (or an "immunogenized" analogue or variant) is herein meant to designate a single polypeptide or protein that includes substantial parts of the sequence information found in native CRIPTO, but preferably does not consist of the entire autologous CRIPTO protein. In one embodiment the CRIPTO analogue is an immunogenic analogue.

"A substantial fragment" of CRIPTO is intended to mean a part of a CRIPTO polypeptide that constitutes at least enough of the monomeric CRIPTO polypeptide so as to form a domain that folds up in substantially the same 3D conformation as can be found in the wildtype protein. In one embodiment, the term, "a substantial fragment" of CRIPTO is refers to a contiguous sequence consisiting of at least 5% of the respective CRIPTO sequence, such as the mature CRIPTO polypeptide sequences or SEQ ID NO 16), such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%. It will be appreciated that a subsequence does not typically refer to a full length sequence - i.e. it consists of less than 100% of the (full or mature) sequence refered to

A "CRIPTO protein" is a functional CRIPTO found in vivo.

An "autologous CRIPTO protein" is a CRIPTO protein which is not foreign to the animal in question, i.e. retains the same primary amino acid sequence as the CRIPTO protein naturally found in said animal, and in one embodiment typically the same (immunogenically speaking) post-translational modifications.

There are numerous CRIPTO protein sequence entries in the NCBI protein database, including : P13385, NP 003203, AAH67844, P51864, AAG49538, and NPJD35692. These sequences, and their corresponding nucleic acid sequences are herby incorporated by reference.

A "CRIPTO polypeptide" is herein intended to denote single-chain polypeptides having an amino acid sequence derived from CRIPTO proteins from humans or other mammals. Unglycosylated forms of CRIPTO, which may be prepared in prokaryotic systems, are included within the boundaries of the term as are forms having varying glycosylation patterns due to the use of e.g. yeasts or other (e.g non-mammalian) eukaryotic expression systems. It should, however, be noted that when using the term "a CRIPTO polypeptide" it is intended that the polypeptide in question is normally non-immunogenic when presented to the animal to be treated. In other words, the CRIPTO polypeptide is a self-molecule or is a xeno- analogue of such a self-molecule which will not normally give rise to an immune response against CRIPTO of the animal in question. However, in one embodiment, within the context of a CRIPTO analogue, which may comprise a CRIPTO polypeptide, the polypeptide may give rise to an immune response against CRIPTO of the animal in question.

A "CRIPTO analogue" is a molecule that includes a CRIPTO polypeptide which has been either subjected to changes in its primary structure and/or that is associated with elements from other molecular species. Such a change can e.g. be in the form of fusion of a CRIPTO polypeptide to a suitable fusion partner {i.e. a change in primary structure exclusively involving C- and/or N-terminal additions of amino acid residues) and/or it can be in the form of insertions and/or deletions and/or substitutions in the CRIPTO polypeptide's amino acid sequence. Also encompassed by the term are derivatized CRIPTO molecules, cf. the discussion below of modifications of CRIPTO.

When using the abbreviation "CRIPTO" herein, this is intended as references to the amino acid sequence of a mature, wildtype CRIPTO (also denoted "CRIPTOm" and "CRIPTOwt". Mature human CRIPTO is denoted hCRIPTO, hCRIPTOm or hCRIPTOwt, and murine mature CRIPTO is denoted mCRIPTO, mCRIPTOm, or mCRIPTOwt. In cases where a DNA construct includes information encoding a leader sequence or other material, this will be clear from the context. The CRIPTO may be the human CRIPTO protein, or a CRIPTO polypeptide or CRITPO analogue derived therefrom.

The CRIPTO polypeptide and/or CRIPTO analogue may, in one embodiment, comprise a contiguous sequence of at least 10 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 40 amino acids, such as at least 50 amino acids, such as at leasδ amino acids, such as at least70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as at least 100 amino acids, such as at least 110 amino acids, such as at least 120 amino acids, such as at least 130 amino acids, which is found in the mature CRIPTO sequence - such as that found in SEQ ID NO 1, SEQ ID NO 16, or the mature CRIPTO sequsence of the CRIPTO sequences present in the NCBI protein database (see above) - or an allelic variant thereof (or suitably a CRIPTO protein which has at least 90%, such as at least 95%, such as at least 96, 97, 98 or 99% homology (identity) to said CRIPTO sequence). In the case of a CRIPTO analogue the contiguous sequence may, in one embodiment, be disrupted by the includion of one or more T_H epitopes, such as those described herein.

The term "polypeptide" is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked. The polypeptide(s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups.

The term "subsequence" means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring CRIPTO amino acid sequence or nucleic acid sequence, respectively. In one embodiment, the term subsequence refers to a contiguous sequence of at least 10 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 40 amino acids, such as at least 50 amino acids, such as at leasδ amino acids, such as at least70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as at least 100 amino acids, such as at least 110 amino acids, such as at least 120 amino acids, such as at least 130 amino acids, which is found in the mature CRIPTO sequence - such as that found in SEQ ID NO 1, SEQ ID NO 16, or the mature CRIPTO sequsence of the CRIPTO sequences present in the NCBI protein database (see above) - or an allelic variant thereof (or suitably a CRIPTO protein which has at least 90%, such as at least 95%, such as at least 96, 97, 98 or 99% homology (identity) to said CRIPTO sequence). In the case of a CRIPTO analogue the contiguous sequence may, in one embodiment, be disrupted by the includion of one or more T_H epitopes, such as those described herein.

In one embodiment, the term subsequence refers to a contiguous sequence of at least 5% of the respective CRIPTO sequence, such as the mature CRIPTO polypeptide sequences, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% of said CRIPTO sequence - such as that found in SEQ ID NO 1, SEQ ID NO 16, or the mature CRIPTO sequence of the CRIPTO sequences present in the NCBI protein database (see above) - or an allelic variant thereof (or suitably a CRIPTO protein which has at least 90%, such as at least 95%, such as at least 96, 97, 98 or 99% homology (identity) to said CRIPTO sequence). . It will be appreciated that a subsequence does not typically refer to a full length sequence - i.e. it consists of less than 100% of the (full or mature) sequence refered to. ). In the case of a CRIPTO analogue the CRIPTO polypeptide sequence may, in one embodiment, be disrupted by the includion of one or more non-CRIPTO sequences, such as one or more T_H epitopes, such as those described herein.

The term "animal" is in the present context in general intended to denote an animal species (preferably mammalian), such as Homo sapiens, Cam^'s domesticus, etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the individuals immunized according to the method of the invention all harbour substantially the same CRIPTO allowing for immunization of the animals with the same immunogen(s). If, for instance, genetic variants of CRIPTO exist in different human populations it may be necessary to use different immunogens in these different populations in order to be able to break the autotolerance towards CRIPTO in each population. It will be clear to the skilled person that an animal in the present context is a living being which has an immune system. It is preferred that the animal is a vertebrate, such as a mammal.

By the term "down-regulation" and "/n vivo down-regulation" is herein meant reduction in the living organism of the biological activity of CRIPTO (e.g. by interference with the interaction between CRIPTO and biologically important binding partners for this molecule). The down- regulation can be obtained by means of several mechanisms: Of these, simple interference with the active site in CRIPTO by antibody binding is the most simple. However, it is also within the scope of the present invention that the antibody binding results in removal of CRIPTO by scavenger cells (such as macrophages and other phagocytic cells). It is also considered that the down-regulation may refer to and/or result in a decrease in the steady state levels of CRIPTO protein and/or mRNA.

The expression "effecting presentation ... to the immune system" is intended to denote that the animal's immune system is subjected to an immunogenic challenge in a controlled manner. As will appear from the disclosure below, such challenge of the immune system can be affected in a number of ways of which the most important are vaccination with polypeptide containing "pharmaccines" (i.e. a vaccine which is administered to treat or ameliorate ongoing disease) or nucleic acid "pharmaccine" vaccination. The important result to achieve is that immune competent cells in the animal are confronted with the antigen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the inventive idea underlying the present invention.

The term "immunogenically effective amount" has its usual meaning in the art, i.e. an amount of an immunogen which is capable of inducing an immune response which significantly engages pathogenic agents which share immunological features with the immunogen.

When using the expression that the CRIPTO has been "modified" is herein meant a chemical modification of the polypeptide which constitutes the backbone of CRIPTO. Such a modification can e.g. be derivatization (e.g. alkylation, acylation, esterification etc.) of certain amino acid residues in the amino acid sequence, but as will be appreciated from the disclosure below, the preferred modifications comprise changes of (or additions to) the primary structure of the amino acid sequence.

When discussing "autotolerance towards CRIPTO" it is understood that since CRIPTO is a self- protein in the population to be vaccinated, normal individuals in the population do not mount an immune response against it; it cannot be excluded, though, that occasional individuals in an animal population might be able to produce antibodies against native CRIPTO, e.g. as part of an autoimmune disorder. At any rate, an animal species will normally only be autotolerant towards its own CRIPTO, but it cannot be excluded that analogues derived from other animal species or from a population having a different phenotype would also be tolerated by said animal.

A "foreign T-cell epitope" (or: "foreign T-lymphocyte epitope") is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species. Preferred foreign T-cell epitopes in the invention are "promiscuous" (or "universal" or "broad-range") epitopes, i.e. epitopes that bind to a substantial fraction of a particular class of MHC molecules in an animal species or population. Only a very limited number of such promiscuous T-cell epitopes are known, and they will be discussed in detail below. It should be noted that in order for the immunogens which are used according to the present invention to be effective in as large a fraction of an animal population as possible, it may be necessary to 1) insert several foreign T-cell epitopes in the same analogue or 2) prepare several analogues wherein each analogue has a different promiscuous epitope inserted. It should be noted also that the concept of foreign T-cell epitopes also encompasses use of cryptic T-cell epitopes, i.e. epitopes which are derived from a self-protein and which only exerts immunogenic behaviour when existing in isolated form without being part of the self-protein in question.

A "foreign T helper lymphocyte epitope" (a foreign T_H epitope) is a foreign T cell epitope which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule. An "MHC Class II binding amino acid sequence that is heterologous to CRIPTO" is therefore an MHC Class II binding peptide that does not exist in CRIPTO. Such a peptide will, if it is also truly foreign to the animal species harbouring CRIPTO, be a foreign T_H epitope.

A "functional part" of a (bio)molecule is in the present context intended to mean the part of the molecule which is responsible for at least one of the biochemical or physiological effects exerted by the molecule. It is well-known in the art that many enzymes and other effector molecules have an active site which is responsible for the effects exerted by the molecule in question. Other parts of the molecule may serve a stabilizing or solubility enhancing purpose and can therefore be left out if these purposes are not of relevance in the context of a certain embodiment of the present invention. However, according to the present invention, it is, inone embodiment, preferred to utilise as much of the polymeric molecule as possible, because this may provide increased biochemical or physiological effects.

The term "adjuvant" has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combination of vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.

"Targeting" of a molecule is in the present context intended to denote the situation where a molecule upon introduction in the animal will appear preferentially in certain tissue(s) or will be preferentially associated with certain cells or cell types. The effect can be accomplished in a number of ways including formulation of the molecule in composition facilitating targeting or by introduction in the molecule of groups which facilitates targeting. These issues will be discussed in detail below.

"Stimulation of the immune system" means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.

The term "substantially specific binding partner" includes in one embodiment the term "specific binding partner".

Characteristics of the immunogenic CRIPTO analogues used in the invention

Although possible, it is not usually desirable to immunize with complete CRIPTO polypeptides or proteins or simple fragments thereof, since this will require formulation with strong adjuvants in order to induce an anti-self CRIPTO immune response. Rather, it is preferred to use an analogue of CRIPTO where at least one modification is present in the CRIPTO amino acid sequence. The modification can have the effect that at least one foreign T helper lymphocyte epitope (T_H epitope) is introduced, and/or that at least one first moiety is introduced which effects targeting of the modified molecule to an antigen presenting cell (APC) or a B-lymphocyte, and/or that at least one second moiety is introduced which stimulates the immune system, and/or that at least one third moiety is introduced which optimizes presentation of the modified CRIPTO polypeptide to the immune system.

Thus, the modification may be introduced as side groups, by covalent or non-covalent binding to suitable chemical groups in the CRIPTO polypeptide or a subsequence thereof, of the foreign T_H epitope and/or of the first and/or of the second and/or of the third moiety, meaning that the moieties or the T_H epitope are fused to or otherwise coupled to or introduced into the CRIPTO polypeptide chain.

Targeting moieties are conveniently selected from the group consisting of a substantially specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen, such as a hapten or a carbohydrate for which there is a receptor on the B- lymphocyte or the APC. The immune stimulating moieties may be selected from the group consisting of a cytokine, a hormone, and a heat-shock protein. The presentation optimising moiety may be selected from the group consisting of a lipid group, such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group.

A suitable cytokine is, or is an effective part of any of, interferon γ (IFN-γ), Flt3L, interleukin 1 (IL-I), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF), and the heat-shock protein is selected from, or is an effective part of any of, HSP70 (heat shock protein 70), HSP90, HSC70 (heat shock cognate 70), GRP94, and calreticulin (CRT).

A preferred heat-shock protein is, or is an effective part of any of, HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT).

Other sequence changes that may enhance immunogenicity is duplication of at least one CRIPTO B-cell epitope and/or introduction of a hapten.

Introduction of the moieties or of the foreign T_H epitopes may include amino acid substitution and/or deletion and/or insertion and/or addition, the latter option providing for a fusion polypeptide.

It is preferred that introduction of the amino acid substitution and/or deletion and/or insertion and/or addition results in a substantial preservation of the overall tertiary structure of the CRIPTO polypeptide , preferably the 3D structure of CRIPTO is essentially preserved.

It is namely advantageous if the immunogenic analogue according to the invention displays, a substantial fraction of B-cell epitopes found in the corresponding CRIPTO protein. A substantial fraction of B-cell epitopes is herein intended to mean a fraction of B-cell epitopes that antigenically characterises the protein versus other proteins and this is best accomplished when the immunogenic analogue is as close in 3D structure to the original native protein as possible.

An especially preferred embodiment provides for an immunogenic analogue of the invention, comprising essentially the complete amino acid sequence of the CRIPTO protein, either as a continuous sequence or as a sequence including inserts. That is, only insignificant parts of the proteins sequence are left out of the analogue, if at all, e.g. in cases where such a sequence does not contribute to tertiary structure of the protein. However, this embodiment allows for substitution or insertion of the protein, as long as the 3D structure of the protein is maintained. Hence, it is especially advantageous if the immunogenic analogue is one, wherein amino acid sequences of the CRIPTO protein are represented in the analogue, and it is particularly advantageous if the analogue includes the complete amino acid sequences of the protein, either as unbroken sequences or as sequences including inserts. As will appear, it is therefore preferred that the 3-dimensional structure of the complete CRIPTO protein is essentially preserved in the analogue.

Demonstration of preservation of a substantial fraction of B-cell epitopes or even the 3- dimensional structure of a CRIPTO protein that is subjected to modification as described herein can be achieved in several ways. One is simply to prepare a polyclonal antiserum directed against native CRIPTO {e.g. an antiserum prepared in a rabbit) and thereafter use this antiserum as a test reagent (e.g. in a competitive ELISA) against the modified proteins which are produced. Modified versions (analogues) which react to the same extent with the antiserum as does the native CRIPTO must be regarded as having the same 3D structure as the native CRIPTO whereas analogues exhibiting a limited (but still significant and specific) reactivity with such an antiserum are regarded as having maintained a substantial fraction of the original B-cell epitopes.

Alternatively, a selection of monoclonal antibodies reactive with distinct epitopes on CRIPTO can be prepared and used as a test panel. This approach has the advantage of allowing 1) an epitope mapping of CRIPTO and 2) a mapping of the epitopes which are maintained in the analogues prepared.

Of course, a third approach would be to resolve the 3-dimensional structure of CRIPTO (cf. above) and compare this to the resolved three-dimensional structure of the analogues prepared. Three-dimensional structure can be resolved by the aid of X-ray diffraction studies and NMR-spectroscopy. Further information relating to the tertiary structure can to some extent be obtained from circular dichroism studies which have the advantage of merely requiring the polypeptide in pure form (whereas X-ray diffraction requires the provision of crystallized polypeptide and NMR requires the provision of isotopic variants of the polypeptide) in order to provide useful information about the tertiary structure of a given molecule. However, ultimately X-ray diffraction and/or NMR are necessary to obtain conclusive data since circular dichroism can only provide indirect evidence of correct 3- dimensional structure via information of secondary structure elements.

The immunogenic analogue of the invention may include a peptide linker that includes or contributes to the presence in the analogue of at least one MHC Class II binding amino acid sequence that is heterologous to the CRIPTO protein. This is particularly useful in those cases where it is undesired to alter the amino acid sequence corresponding to CRIPTO. Alternatively, the peptide linker may be free of and not contributing to the presence of an MHC Class II binding amino acid sequence in the animal species from where the CRIPTO protein is derived; this can conveniently be done in cases where it is necessary to utilise a very short linker or where it is advantageous to e.g. detoxify a potentially toxic analogue by introducing the MHC Class II binding element in an active site.

Both these embodiments can be combined with introduction of point mutations that detoxify (or render inactive) the molecule if need be, cf. below.

In other embodiments, no peptide linker is included, and in these cases the introduction of an MHC Class II binding amino acid sequence is performed by means of insertion, addition, deletion or substitution in the CRIPTO polypeptide sequence.

It is preferred that the MHC Class II binding amino acid sequence binds a majority of MHC Class II molecules from the animal species from where the CRIPTO protein has been derived, i.e. that the MHC Class II binding amino acid sequence is universal or promiscuous.

It is of course important that this sequence serves its purpose as a T helper cell epitope in the species for which the immunogen is intended to serve as a vaccine constituent. There exists a number of naturally occurring "promiscuous" (or "universal") T-cell epitopes which are active in a large proportion of individuals of an animal species or an animal population and these are preferably introduced in the vaccine, thereby reducing the need for a very large number of different analogues in the same vaccine. Hence, at least one MHC Class II binding amino acid sequence is preferably selected from a natural T-cell epitope and an artificial MHC-II binding peptide sequence. Especially preferred sequences are a natural T-cell epitope selected from a Tetanus toxoid epitope such as P2 (SEQ ID NO: 26) or P30 (SEQ ID NO: 27), a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope.

Over the years a number of other promiscuous T-cell epitopes have been identified. Especially peptides capable of binding a large proportion of HLA-DR molecules encoded by the different HLA-DR alleles have been identified and these are all possible T-cell epitopes to be introduced in the analogues used according to the present invention. Cf. also the epitopes discussed in the following references which are hereby all incorporated by reference herein: WO 98/23635 (Frazer IH ef al., assigned to The University of Queensland); Southwood S et. al, 1998, J. Immunol. 160: 3363-3373; Sinigaglia F et al., 1988, Nature 336: 778-780; Chicz RM et al., 1993, J. Exp. Med 178: 27-47; Hammer J et al., 1993, Cell 74: 197-203; and FaIk K et al., 1994, Immunogenetics 39: 230-242. The latter reference also deals with HLA-DQ and -DP ligands. All epitopes listed in these 5 references are relevant as candidate natural epitopes to be used in the present invention, as are epitopes that share common motifs with these.

Alternatively, the epitope can be any artificial T-cell epitope which is capable of binding a large proportion of MHC Class II molecules. In this context the pan DR epitope peptides ("PADRE") described in WO 95/07707 and in the corresponding paper Alexander J ef a/., 1994, Immunity 1: 751-761 (both disclosures are incorporated by reference herein) are interesting candidates for epitopes to be used according to the present invention. It should be noted that the most effective PADRE peptides disclosed in these papers carry D-amino acids in the C- and N-termini in order to improve stability when administered. However, the present invention primarily aims at incorporating the relevant epitopes as part of the analogue which should then subsequently be broken down enzymatically inside the lysosomal compartment of APCs to allow subsequent presentation in the context of an MHC-II molecule and therefore it is not expedient to incorporate D-amino acids in the epitopes used in the present invention.

One especially preferred PADRE peptide is the one having the amino acid sequence

AKFVAAWTLKAAA (SEQ ID NO:21) or an immunologically effective subsequence thereof. This, and other epitopes having the same lack of MHC restriction are preferred T-cell epitopes which should be present in the analogues used in the inventive method. Such super- promiscuous epitopes will allow for the most simple embodiments of the invention wherein only one single modified CRIPTO is presented to the vaccinated animal's immune system.

As mentioned above, the introduction of a foreign T-cell epitope can be accomplished by introduction of at least one amino acid insertion, addition, deletion, or substitution. Of course, the normal situation will be the introduction of more than one change in the amino acid sequence (e.g. insertion of or substitution by a complete T-cell epitope) but the important goal to reach is that the analogue, when processed by an antigen presenting cell (APC), will give rise to such a T-cell epitope being presented in context of an MCH Class II molecule on the surface of the APC. Thus, if the amino acid sequence of the monomeric unit in appropriate positions comprises a number of amino acid residues which can also be found in a foreign T_H epitope then the introduction of a foreign T_H epitope can be accomplished by providing the remaining amino acids of the foreign epitope by means of amino acid insertion, addition, deletion and substitution. In such a situation, it is not necessary to introduce a complete T_H epitope by insertion or substitution. It is preferred that the number of amino acid insertions, deletions, substitutions or additions is at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29 or 30 insertions, substitutions, additions or deletions, such as between 10 and 30 insertions, substitutions, additions or deletions. It is furthermore preferred that the number of amino acid insertions, substitutions, additions or deletions is not in excess of 150, such as at most 100, at most 90, at most 80, and at most 70. It is especially preferred that the number of substitutions, insertions, deletions, or additions does not exceed 60, and in particular the number should not exceed 50 or even 40. Most preferred is a number of not more than 30. With respect to amino acid additions, it should be noted however, that these, when the resulting construct is in the form of a fusion polypeptide, is often considerably higher than 150.

Preferred embodiments of the invention includes modification by introducing at least one foreign immunodominant T_H epitope (= "foreign MHC Class II binding amino acid sequence"). It will be understood that the question of immune dominance of a T_H epitope depends on the animal species in question. As used herein, the term "immunodominance" simply refers to epitopes which in the vaccinated individual gives rise to a significant immune response, but it is a well-known fact that a T_H epitope which is immunodominant in one individual is not necessarily immunodominant in another individual of the same species, even though it may be capable of binding MHC-II molecules in the latter individual.

An important point is the issue of MHC restriction of T_H epitopes. In general, naturally occurring T_H epitopes are MHC restricted, i.e. a certain peptide constituting a T_H epitope will only bind effectively to a subset of MHC Class II molecules. This in turn has the effect that in most cases the use of one specific T_H epitope will result in a vaccine component which is effective in a fraction of the population only, and depending on the size of that fraction, it can be necessary to include more T_H epitopes in the same molecule, or alternatively prepare a multi-component vaccine wherein the components are variants which are distinguished from each other by the nature of the T_H epitope introduced.

If the MHC restriction of the T-cells used is completely unknown (for instance in a situation where the vaccinated animal has a poorly defined MHC composition), the fraction of the animal population covered by a specific vaccine composition can be determined by means of the following formula:

J population X X ^ * ι ) (I) i=l -where p, is the frequency in the population of responders to the /^th foreign T-cell epitope present in the vaccine composition, and n is the total number of foreign T-cell epitopes in the vaccine composition. Thus, a vaccine composition containing 3 foreign T-cell epitopes having response frequencies in the population of 0.8, 0.7, and 0.6, respectively, would give

1 - 0.2 x 0.3 x 0.4 = 0.976

-i.e. 97.6 percent of the population will statistically mount an MHC-II mediated response to the vaccine.

The above formula does not apply in situations where a more or less precise MHC restriction pattern of the peptides used is known. If, for instance a certain peptide only binds the human MHC-II molecules encoded by HLA-DR alleles DRl, DR3, DR5, and DR7, then the use of this peptide together with another peptide which binds the remaining MHC-II molecules encoded by HLA-DR alleles will accomplish 100% coverage in the population in question. Likewise, if the second peptide only binds DR3 and DR5, the addition of this peptide will not increase the coverage at all. If one bases the calculation of population response purely on MHC restriction of T-cell epitopes in the vaccine, the fraction of the population covered by a specific vaccine composition can be determined by means of the following formula:

-wherein φ_} is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind any one of the T-cell epitopes in the vaccine and which belong to the /^h of the 3 known HLA loci (DP, DR and DQ); in practice, it is first determined which MHC molecules will recognize each T-cell epitope in the vaccine and thereafter these MHC molecules are listed by type (DP, DR and DQ) - then, the individual frequencies of the different listed allelic haplotypes are summed for each type, thereby yielding φ_lr <fø, and φ₃.

It may occur that the value p, in formula I exceeds the corresponding theoretical value π,:

-wherein v, is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind the F^h T-cell epitope in the vaccine and which belong to the /^h of the 3 known HLA loci (DP, DR and DQ). This means that in 1-π, of the population there is a frequency of responders of f_resιduau = (Prπ,)/(l-π,). Therefore, formula II can be adjusted so as to yield formula V:

(^IV)

-where the term l-f_reSιduai_/ is set to zero if negative. It should be noted that formula IV requires that all epitopes have been haplotype mapped against identical sets of haplotypes.

Therefore, when selecting T-cell epitopes to be introduced in the analogue of the invention, it is important to include all knowledge of the epitopes which is available: 1) The frequency of responders in the population to each epitope, 2) MHC restriction data, and 3) frequency in the population of the relevant haplotypes.

It should be noted that preferred analogues of the invention comprise modifications which results in a polypeptide that includes stretches having a sequence identity of at least 70% with the corresponding monomeric units of the CRIPTO protein or with subsequences thereof of at least 10 amino acids in length. Higher sequence identities are preferred, e.g. at least 75% or even at least 80% or at least 85% or such as at least 90%, The sequence identity for proteins and nucleic acids can be calculated as {N_ref - N_dlf)-100/N_refl wherein N_dlf is the total number of non-identical residues in the two sequences when aligned and wherein N_ref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (ΛW=2 and N_ref=8).

Finally, in order to conclusively verify that an analogue of the invention is indeed effective as an immunogen, various tests may be performed in order to provide the necessary confirmation, cf. also the specifics set forth in the examples herein. In this context, reference is also made to the discussion of identification of useful IL5 analogues in WO 00/65058 - this disclosure may be used for verification of the usefulness of a CRIPTO analogue subject to the present inventive technology.

It is preferred that the autologous CRIPTO polypeptide is a human CRIPTO - polypeptide, preferably SEQ ID NO: 16, and naturally occurring allelic variants thereof. Known natural allelic variants of CRIPTO include V22A and Y43D. Naturally occurring variants of the CRIPTO sequence may typically comprise between 1 to 10 point mutations, wuch as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 point mutations. They may also comprise deletions, substitutions or insertions. The CRIPTO polypeptide may consisit of a non-naturally occurring CRIPTO sequence, such as recombinantly modified CRIPTO polypeptides.

For the preparation of CRIPTO analogues such as immunogenic analogues, autologous CRIPTO polypeptides may be used. Alternatively, a CRIPTO polypeptide wherein is introduced at least one modification in the CRIPTO amino acid sequence, such as SEQ ID No 1.

The cloned wild type (wt) human CRIPTO sequence has 188 amino-acid residues and a calculated molecular weight of 36 kDa with fucosylations and glycosylations and 21 kDa without glycosylations¹⁹.

It is well known that each of the EGF-like and CFC domains have a much conserved disulfide bond pattern with three disulfide bridges in each domain. By peptide-mapping it has been shown that these six disulfide bonds are located in human CRIPTO at positions: Cys82- Cys89; Cys83-Cys95; Cys97-CyslO6; Cysll5-Cysl33; Cysl28-Cysl49 and Cysl31-Cysl40. Furthermore, there are two free cystein residues in the terminal ends: Cys3 and Cyslβl. The degree of standard structure elements like alpha-helixes and beta-sheets in CRIPTO is limited. The molecule is held together by the six disulfide-bridges. Some structure transformation could occur after binding of CRIPTO to its ligand(s). In one embodiment the CRIPTO analogue according to the invention retains the six disulphide bridges found in the human CRIPTO protein.

As will appear from the examples, one preferred analogue is a human CRIPTO polypeptide which consists of 140 residues starting at Leu31 of cloned CRIPTO sequence (SEQ ID No 16) and ending at Alal70 of (SEQ ID No 16), represented as SEQ ID No 1, with, optionally further insertions, deletions and/or substitutions. SEQ ID No 1 can therefore be used as a template for the preparation of further CRIPTO analogues such as immunogenic analogues. The carboxyl terminal of SEQ ID No 1 is in the middle of the GPI anchor sequence.

In one embodiment, the CRIPTO analogues have a disrupted GPI anchor sequence, such as deletion, insertion or substitution within the GPI anchor sequence, including, as shown is SEQ ID No 1, a carboxy terminal truncation within the GPI anchor sequence.

Therefore, SEQ ID No 16 or SEQ ID No 1 may, for example, be used as templates for the preparation of CRIPTO analogues.

Preferable regions for the insertion of a foreign T cell epitope in human CRIPTO are localized in the areas outside of the conserved EGF abd CFC domains. The preferred regions are predicted to have predominantly random coil structures. Suitable preferred regions, based on the amino acid positions of the hCRwt sequence (SEQ ID No 1), include:

Leul-Pro39 (referred to herein as hCRl AutoVac™ molecules) : hCRl CRIPTO analogues prepared by the insertion of the foreign T cell epitope PADRE include SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, and SEQ ID No 5. Specific insertion sites for insertion of foreign T cell epitopes into the CRIPTO sequence therefore Ala7-Arg8, Alal5-Phel6, Ile21-Trp22, Pro27- Ile29. Thus, especially preferred constructs are those wherein the human CRIPTO polypeptide has been modified by insertion into, deletion in, addition to, or substitution of any one of amino acids 1-39 in SEQ ID NO 1. This means that preferred constructs entail insertion after any one of amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 and/or deletion or substitution of any one of amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 in SEQ ID NO 1.

Met40-Thr51 (referred to herein as hCR2 AutoVac™ molecules) : hCR2 CRIPTO analogues prepared by the insertion of the foreign T cell epitope PADRE include SEQ ID No 6 and SEQ ID No 7. Specific insertion sites for insertion of foreign T cell epitopes into the CRIPTO sequence therefore Gly41-Ile42, and Leu48-Asn49. Thus, especially preferred constructs are those wherein the human CRIPTO polypeptide has been modified by insertion into, deletion in, addition to, or substitution of any one of amino acids 40-51 in SEQ ID NO 1. This means that preferred constructs entail insertion after any one of amino acids 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, and 51 and/or deletion or substitution of any one of amino acids 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, and 51 of SEQ ID NO 1.

Lys82-Asn84 (referred to herein as hCR3 AutoVac™ molecules) : hCR3 CRIPTO analogues prepared by the insertion of the foreign T cell epitope PADRE include SEQ ID No 8, SEQ ID No 9 and SEQ ID No 10. Specific insertion sites for insertion of foreign T cell epitopes into the

CRIPTO sequence therefore Lys82-Glu83, Glu83-Asn84, and Asn84-Gly85. The insertion may also be accompanied by a duplication of the linker region or parts thereof corresponding to amino acids Arg81-Cys85, as exemplified by SEQ ID No 10. Thus, especially preferred constructs are those wherein the human CRIPTO polypeptide has been modified by insertion into, deletion in, addition to, or substitution of any one of amino acids 82-84 in SEQ ID NO 1. This means that preferred constructs entail insertion after any one of amino acids 81, 82, 83 and 84 and/or deletion or substitution of any one of amino acids 81, 82, 83 and 84 of SEQ ID NO 1.

Glyl21-Alal40 (referred to herein as hCR4 AutoVac™ molecules) : hCR4 CRIPTO analogues prepared by the insertion of the foreign T cell epitope PADRE include SEQ ID No 11, SEQ ID No 12 and SEQ ID No 13. Specific insertion sites for insertion of foreign T cell epitopes into the CRIPTO sequence therefore Vall29-Alal30, Glul35-Leul36, and Serl39-Alal40. Thus, especially preferred constructs are those wherein the human CRIPTO polypeptide has been modified by insertion into, deletion in, addition to, or substitution of any one of amino acids 121-140 in SEQ ID NO 1. This means that preferred constructs entail insertion after any one of amino acids 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 and 140 and/or deletion or substitution of any one of amino acids 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 and 140 of SEQ ID NO 1.

In one embodiment, the CRIPTO analogue may be a single domain of the CRIPTO incorporating, or fused to a foreign T cell epitope. An example of an isolated EGF domain fused to the PADRE sequence is shown as SEQ ID Nol4. An example of an isolated CFC domain fused to the PADRE sequence is shown as SEQ ID No 15. Suitable single domains include the EGF domain (SEQ ID No 22), and the CFC domain (SEQ ID No 23). In one embodiment fragments of such single domains may also be used as long as the fragment comprises at least one B cell epitope. In one embodiment, the single domain epitopes may also comprise a short region of sequence flanking said single domain, such as between 1 and 30 amino acids, including between 1 and 20 and between 1 and 10 amino acids.

It is envisaged that any fragment of CRIPTO may be used as a template for the preparation of a CRIPTO analogue as long as the fragment (and/or analogue preprared therefrom) comprises at least one B cell epitope which is present in the autologous CRIPTO protein.

In one embodiment, the CRIPTO template and/or analogue prepared therefrom comprises a mutation, such as an insertion, substitution and/or deletion, preferably a point mutation (substitution), which destroys a proteolytic cleavage site within the CRIPTO sequence, thereby improving the in vivo stability of the CRIPTO analogue. The enhanced stability may be seen when expressed in a heterologous host cell, and/or when used as a protein vaccine. One preferred point mutation is a point mutation at position Arg32, substituting the Arg32 residue with a residue which is more hydrophobic, such as valine {i.e. Arg32-Val32 substitution). Other hydrophobic residues which could be used include, for example an amino acid selected from the group comprising GIy, Ala, Pro, He or Leu). Such substitutions may occour not only at position Arg32, but may, for example also occur at any position within the first 50 residues of the N-terminus of SEQ ID No 1. In one embodiment the CRIPTO template and/or analogue prepared therefrom comprises a N-terminal truncation when compared to the hCRwt sequence (SEQ ID No 1). Suitable the N terminal trucations may be a truncation which occurs between amino acids 1 and 50 of the hCRwt sequence, such as a truncation between amino acid 1, and the amino acid at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 of the hCRWT sequence. Preferred truncations include truncation between amino acid 1 and an amino acids selected form the group consisting of amino acids 30,31, 32, 33, 34, and 35.

N-terminal truncations may be introduced by the introduction of a Furin cleavage site by mutation of the genetic code encoding the CRIPTO template and/or anologue prepared thereform. In this way, if the deletion of the N-terminus is found to prevent or hinder secretion in an expression host due to failed entry into the ER, the N-terminus may be cleaved after entry into the ER and prior to secretion.

C-terminal truncations may also be introduced, for example the C terminal region may be deleted after the CFC domain, for example as shown in SEQ ID No 99 and SEQ ID No 100 (which also comprises a R32V substitution). Such trucations may be made at or around G121 of SEQ ID No 1 (in this context "or around" means within 20 residues, such as within 10 residues, such as within 5 residues, such as within 2 residues of G121 or any subsequent residue of SEQ ID No 1 - in either the 5' or 3' direction, or both 5' and 3' directions). Such deletions may improve protein solubility. The CRIPTO analogue according to the invention may therefore comprise a R32V substitution. Suitably C terminal truncations may be made at (or around) residue 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139 or residue 140 of SEQ ID No 1, when compared to the wildtype human CRITPO protein.

Design of His-tagged CRIPTO AutoVac™ molecules

During the past few years, a number of purification tag systems have been developed to facilitate and standardize purification of recombinant proteins ²²'²³. In these systems, a terminal polypeptide forms a protein tag with binding specificity suitable for affinity purification and is fused to the protein of interest, most frequently to the N-terminus.

The addition of a histidine-rich peptide tag (polyhistidine tag; HisTag) to the target protein is a simple and well-established approach for generating a novel binding specificity. This makes one-step purification possible when using immobilized metal affinity chromatography (IMAC). IMAC matrices hold a number of advantages including high protein binding capacity and ligand stability, low costs and the use of mild elution conditions. Furthermore, because of their chemical nature, IMAC matrices can easily be sanitized and regenerated making them suitable for large-scale applications.

In case purification of non-tagged CRIPTO AutoVac™ proteins is problematic, in order to facilitate down stream processing from an expression host, it is preferred that the CRIPTO protein (such as the autologous CRIPTO protein), CRIPTO polypeptide/template and/or anologue prepared therefrom comprises a purification tag, such as a His-tag. A preferred HisTag for this purpose is the UniHisTag - with the sequence MKHQHQHQHQHQHQAP (SEQ ID No 98). In one embodiment, the HisTag may be fused to the C-terminus of the CRIPTO molecule.

Following elution of the His-tagged CRIPTO protein from an IMAC column, the HisTag can be cleaved using the TAGZyme System developed by Unizymes. The TAGZyme System is an enzymatic system for the complete removal of N-terminal poly-histidine tags from recombinant proteins using the dipeptidyl amino-peptidase I (DAPase) enzyme which catalyzes the stepwise removal of N-terminal dipeptides except if (A) the amino group of the N-terminus is blocked, (B) the site of cleavage is on either side of a proline, or (C) the N- terminal residue is either lysine or arginine. The UniHisTag does not contain lysine or arginine and the proline (P16) stops the cleavage reaction giving the same N-terminal sequence of all the cleaved protein. Following cleavage with DAPase a subtractive IMAC will bind the cleaved HisTag as well as the DAPase enzyme (also containing a HisTag) rendering the CRIPTO protein ready for further downstream purification.

Further constructs entail insertion after any one of amino acids 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 and 120 of SEQ ID No 1, and/or deletion or substitution of any one of amino acids 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 and 120 of SEQ ID NO 1.

In one embodiment, two or more CRIPTO sequences, such as CRIPTO proteins, polypeptides or analogues may be fused or otherwise joined together. For example, a single polypeptide chain containing two CRIPTO sequences may be created by the fusion of the two polypeptises to a spacer sequence, such as a short glycine linker. Such polymerisation of the CRIPTO polypeptides can result in improved stability of the analogue produced.

The linker sequence used to join two or more CRIPTO sequences may itself comprise a foreign T-cell epitope. Therefore, in one embodiment, the CRIPTO vaccine according to the invention is prepared by fusing two or more CRIPTO sequences to a linker sequence which comprises one or more foreign T-cell epitope.

The polymerisation may occur between two CRIPTO proteins, or fragments thereof, which may subsequently be used to prepare CRIPTO vaccines according to the invention.

The polypermisation may be performed between a CRIPTO protein and a CRIPTO analogue, such as a CRIPTO analogue which comprises a foreign T-cell epitope.

The polymerisation may be performed between two CRIPTO analogues, which may be the same or different. Protein/polypeptide vaccination and formulation

When effecting presentation of the analogues to an animal's immune system by means of administration thereof to the animal, the formulation of the polypeptide follows the principles generally acknowledged in the art.

Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.

The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutaneously, intradermally, subdermally or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral, buccal, sublinqual, intraperitoneal, intravaginal, anal, epidural, spinal, and intracranial formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%. For oral formulations, cholera toxin is an interesting formulation partner (and also a possible conjugation partner).

The polypeptides may be formulated into the vaccine as neutral or salt forms.

Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 2,000 μg (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 μg to 1,000 μg, preferably in the range from 1 μg to 500 μg and especially in the range from about 10 μg to 100 μg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and the formulation of the antigen.

In order to enhance immunogenicity of a polypeptide construct of the invention, it can be ensured that presentation to the immune system is effected by having at least two copies of the CRIPTO polypeptide, the subsequence thereof or the modified CRIPTO polypeptide covalently of non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants. Such carriers may be polysaccharides or any other polymer substance capable of presenting polypeptides.

Some of the analogues of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effect for the vaccine are known. General principles and methods are detailed in "The Theory and Practical Application of Adjuvants", 1995, Duncan E. S. Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in "Vaccines: New Generation Immunological Adjuvants", 1995, Gregoriadis G et al. (eds.)_/ Plenum Press, New York, ISBN 0-306-45283-9, both of which are hereby incorporated by reference herein.

It is especially preferred to use an adjuvant which can be demonstrated to facilitate breaking of the autotolerance to autoantigens; in fact, this is essential in cases where unmodified CRIPTO is used as the active ingredient in the autovaccine. Non-limiting examples of suitable adjuvants are selected from the group consisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ- inulin; and an encapsulating adjuvant. In general it should be noted that the disclosures above which relate to compounds and agents useful as first, second and third moieties in the analogues also refer mutatis mutandis to their use in the adjuvant of a vaccine of the invention.

The application of adjuvants include use of agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101⁰C for 30 second to 2 minute periods respectively and also aggregation by means of cross-linking agents are possible. Aggregation by reactivation with pepsin treated antibodies (Fab fragments) to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Admixture with oils such as squalene and IFA is also preferred.

According to the invention DDA (dimethyldioctadecylammonium bromide) is an interesting candidate for an adjuvant as is DNA and γ-inulin, but also Freund's complete and incomplete adjuvants as well as quillaja saponins such as QuilA and QS21 are interesting as is RIBI. Further possibilities are monophosphoryl lipid A (MPL), the above mentioned C3 and C3d, and muramyl dipeptide. One preferred adjuvant is Provax® (Biogen).

Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants are preferred according to the invention. Also immunostimulating complex matrix type (ISCOM® matrix) adjuvants are preferred choices according to the invention, especially since it has been shown that this type of adjuvants are capable of up-regulating MHC Class II expression by APCs. An ISCOM® matrix consists of (optionally fractionated) saponins (triterpenoids) from Quillaja saponaria, cholesterol, and phospholipid. When admixed with the immunogenic protein, the resulting particulate formulation is what is known as an ISCOM particle where the saponin constitutes 60-70% w/w, the cholesterol and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details relating to composition and use of immunostimulating complexes can e.g. be found in the above-mentioned text-books dealing with adjuvants, but also Morein B et al., 1995, Clin. Immunother. 3: 461-475 as well as Barr IG and Mitchell GF, 1996, Immunol, and Cell Biol. 74: 8-25 (both incorporated by reference herein) provide useful instructions for the preparation of complete immunostimulating complexes.

Another highly interesting (and thus, preferred) possibility of achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference herein). In brief, the presentation of a relevant antigen such as an antigen of the present invention can be enhanced by conjugating the antigen to antibodies (or antigen binding antibody fragments) against the Fcγ receptors on monocytes/macrophages. Especially conjugates between antigen and anti-FcγRI have been demonstrated to enhance immunogenicity for the purposes of vaccination.

Other possibilities involve the use of the targeting and immune modulating substances (J. a. cytokines) mentioned in the claims as moieties for the protein constructs. In this connection, also synthetic inducers of cytokines like poly I:C are possibilities.

Suitable mycobacterial derivatives are selected from the group consisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, and a diester of trehalose such as TDM and TDE.

Suitable immune targeting adjuvants are selected from the group consisting of CD40 ligand and CD40 antibodies or specifically binding fragments thereof (cf. the discussion above), mannose, a Fab fragment, and CTLA-4.

Suitable polymer adjuvants are selected from the group consisting of a carbohydrate such as dextran, PEG, starch, mannan, and mannose; a plastic polymer such as; and latex such as latex beads. Yet another interesting way of modulating an immune response is to include the immunogen (optionally together with adjuvants and pharmaceutically acceptable carriers and vehicles) in a "virtual lymph node" (VLN) (a proprietary medical device developed by ImmunoTherapy, Inc., 360 Lexington Avenue, New York, NY 10017-6501). The VLN (a thin tubular device) mimics the structure and function of a lymph node. Insertion of a VLN under the skin creates a site of sterile inflammation with an upsurge of cytokines and chemokines. T- and B-cells as well as APCs rapidly respond to the danger signals, home to the inflamed site and accumulate inside the porous matrix of the VLN. It has been shown that the necessary antigen dose required to mount an immune response to an antigen is reduced when using the VLN and that immune protection conferred by vaccination using a VLN surpassed conventional immunization using Ribi as an adjuvant. The technology is La. described briefly in Gelber C ef a/., 1998, "Elicitation of Robust Cellular and Humoral Immune Responses to Small Amounts of Immunogens Using a Novel Medical Device Designated the Virtual Lymph Node", in: "From the Laboratory to the Clinic, Book of Abstracts, October 12^th - 15^th 1998, Seascape Resort, Aptos, California".

It is expected that the vaccine should be administered at least once a year, such as at least 1, 2, 3, 4, 5, 6, and 12 times a year. More specifically, 1-12 times per year is expected, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times a year to an individual in need thereof. It has previously been shown that the memory immunity induced by the use of the preferred autovaccines according to the invention is not permanent, and therefore the immune system needs to be periodically challenged with the analogues.

Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response, cf. also the discussion above concerning the choice of foreign T-cell epitope introductions. The vaccine may comprise two or more polypeptides, where all of the polypeptides are as defined above.

The vaccine may consequently comprise 3-20 different analogues, such as 3-10 analogues. However, normally the number of analogues will be sought kept to a minimum such as 1 or 2 analogues.

Nucleic acid vaccination As a very important alternative to classic administration of a peptide-based vaccine, the technology of nucleic acid vaccination (also known as "nucleic acid immunisation", "genetic immunisation", and "gene immunisation") offers a number of attractive features.

First, in contrast to the traditional vaccine approach, nucleic acid vaccination does not require resource consuming large-scale production of the immunogenic agent (e.g. in the form of industrial scale fermentation of microorganisms producing proteins). Furthermore, there is no need for device purification and refolding schemes for the immunogen. And finally, since nucleic acid vaccination relies on the biochemical apparatus of the vaccinated individual in order to produce the expression product of the nucleic acid introduced, the optimum posttranslational processing of the expression product is expected to occur; this is especially important in the case of autovaccination, since, as mentioned above, a significant fraction of the original B-cell epitopes of the polymer should be preserved in the modified molecule, and since B-cell epitopes in principle can be constituted by parts of any (bio)molecule (e.g. carbohydrate, lipid, protein etc.). Therefore, native glycosylation and lipidation patterns of the immunogen may very well be of importance for the overall immunogenicity and this is expected to be ensured by having the host producing the immunogen.

It should be noted that the enhanced expression levels observed with the presently disclosed analogues is very important for efficacy of DNA vaccination, since the in vivo expression level is one of the determining factors in the immunogenic efficacy of a DNA vaccine

Hence, a preferred embodiment of the invention comprises effecting presentation of the analogue of the invention to the immune system by introducing nucleic acid(s) encoding the analogue into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced.

In this embodiment, the introduced nucleic acid is preferably DNA which can be in the form of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. the microencapsulation technology described in WO 98/31398) or in chitin or chitosan, and DNA formulated with an adjuvant. In this context it is noted that practically all considerations pertaining to the use of adjuvants in traditional vaccine formulation apply for the formulation of DNA vaccines. Hence, all disclosures herein which relate to use of adjuvants in the context of polypeptide based vaccines apply mutatis mutandis to their use in nucleic acid vaccination technology.

As for routes of administration and administration schemes of polypeptide based vaccines which have been detailed above, these are also applicable for the nucleic acid vaccines of the invention and all discussions above pertaining to routes of administration and administration schemes for polypeptides apply mutatis mutandis to nucleic acids. To this should be added that nucleic acid vaccines can suitably be administered intraveneously and intraarterially. Furthermore, it is well-known in the art that nucleic acid vaccines can be administered by use of a so-called gene gun, and hence also this and equivalent modes of administration are regarded as part of the present invention. Finally, also the use of a VLN in the administration of nucleic acids has been reported to yield good results, and therefore this particular mode of administration is particularly preferred.

Furthermore, the nucleic acid(s) used as an immunization agent can contain regions encoding the moieties specified in the claims, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitope is produced as a fusion partner to the immunomodulator. Alternatively, two distinct nucleotide fragments can be used, but this is less preferred because of the advantage of ensured co-expression when having both coding regions included in the same molecule.

Accordingly, the invention also relates to a composition for inducing production of antibodies against CRIPTO, the composition comprising

a nucleic acid fragment or a vector of the invention (cf. the discussion of nucleic acids and vectors below), and

a pharmaceutically and immunologically acceptable vehicle and/or carrier and/or adjuvant as discussed above.

Under normal circumstances, the nucleic acid is introduced in the form of a vector wherein expression is under control of a viral promoter. For more detailed discussions of vectors and DNA fragments according to the invention, cf. the discussion below. Also, detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly JJ et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly JJ et al., 1997, Life Sciences 60: 163- 172. Both of these references are incorporated by reference herein.

Live vaccines

A third alternative for effecting presentation of the analogues of the invention to the immune system is the use of live vaccine technology. In live vaccination, presentation to the immune system is effected by administering, to the animal, a non-pathogenic microorganism that has been transformed with a nucleic acid fragment encoding an analogue of the invention or with a vector incorporating such a nucleic acid fragment. The non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by means of passaging or by means of removal of pathogenic expression products by recombinant DNA technology), e.g.

Mycobacterium bovis BCG., non-pathogenic Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae, Shigella, etc. Reviews dealing with preparation of state-of-the-art live vaccines can e.g. be found in Saliou P, 1995, Rev. Prat. 45: 1492-1496 and Walker PD, 1992, Vaccine 10: 977-990, both incorporated by reference herein. For details about the nucleic acid fragments and vectors used in such live vaccines, cf. the discussion below.

As an alternative to bacterial live vaccines, the nucleic acid fragment of the invention discussed below can be incorporated in a non-virulent viral vaccine vector such as a vaccinia strain or any other suitable pox virus.

Normally, the non-pathogenic microorganism or virus is administered only once to the animal, but in certain cases it may be necessary to administer the microorganism more than once in a lifetime in order to maintain protective immunity. It is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.

Alternatively, live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination. For instance, it is possible to effect primary immunization with a live or virus vaccine followed by subsequent booster immunizations using the polypeptide or nucleic acid approach.

The microorganism or virus can be transformed with nucleic acid(s) containing regions encoding the moieties mentioned above, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitopes are produced as fusion partners to the immunomodulator. Alternatively, two distinct nucleotide fragments can be used as transforming agents. Of course, having the adjuvating moieties in the same reading frame can provide, as an expression product, an analogue of the invention, and such an embodiment is especially preferred according to the present invention.

Combination treatment

One especially preferred mode of carrying out the invention involves the use of nucleic acid vaccination as the first (primary) immunization, followed by secondary (booster) immunizations with a polypeptide based vaccine or a live vaccine as described above.

Use of the method of the invention in treatment of specific diseases

All solid tumors rely on angiogenesis for their growth and metastatic properties while normal vasculature is quiescent in healthy adults, with each endothelial cell dividing once every 10 years. Angiogenesis provides then an attractive therapeutic target for therapy of solid tumours and with a theoretically limited toxicity profile.

Combined with conventional cytotoxic agents, anti-CRIPTO antibody therapy demonstrated potent anti-tumor activity in different cancers including breast, pancreatic, colon, lung, and ovarian cancers.

Altogether, these findings demonstrate the broad applicability of CRIPTO immunotherapy among solid and haematological tumor indications.

Compositions of the invention

The invention also pertains to compositions useful in exercising the method of the invention. Hence, the invention also relates to an immunogenic composition comprising an immunogenically effective amount of an analogue defined above, said composition further comprising a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or carrier and/or excipient and optionally an adjuvant. In other words, this part of the invention concerns formulations of analogues, essentially as described hereinabove. The choice of adjuvants, carriers, and vehicles is accordingly in line with what has been discussed above when referring to formulation of the analogues for peptide vaccination. The analogues are prepared according to methods well-known in the art. Longer polypeptides are normally prepared by means of recombinant gene technology including introduction of a nucleic acid sequence encoding the analogue into a suitable vector, transformation of a suitable host cell with the vector, expression of the nucleic acid sequence (by culturing the host cell under appropriate conditions), recovery of the expression product from the host cells or their culture supernatant, and subsequent purification and optional further modification, e.g. refolding or derivatization. Details pertaining to the necessary tools are found below under the heading "Nucleic acid fragments and vectors of the invention" but also in the examples.

Shorter peptides are, when relevant, preferably prepared by means of the well-known techniques of solid- or liquid-phase peptide synthesis. However, recent advances in this technology has rendered possible the production of full-length polypeptides and proteins by these means, and therefore it is also within the scope of the present invention to prepare the long constructs by synthetic means.

Nucleic acid fragments and vectors of the invention

It will be appreciated from the above disclosure that modified polypeptides can be prepared by means of recombinant gene technology but also by means of chemical synthesis or semi- synthesis; the latter two options are especially relevant when the modification consists of or comprises coupling to protein carriers (such as KLH, diphtheria toxoid, tetanus toxoid, and BSA) and non-proteinaceous molecules such as carbohydrate polymers and of course also when the modification comprises addition of side chains or side groups to an polymer-derived peptide chain. These embodiments, are, as will be understood from the above, not the preferred ones.

For the purpose of recombinant gene technology, and of course also for the purpose of nucleic acid immunization, nucleic acid fragments encoding the analogues are important chemical products (as are their complementary sequences). Hence, an important part of the invention pertains to a nucleic acid fragment which encodes an analogue as described herein, i.e. a polymer derived artificial polymer polypeptide as described in detail above. The nucleic acid fragments of the invention are either DNA or RNA fragments.

The nucleic acid fragments of the invention will normally be inserted in suitable vectors to form cloning or expression vectors carrying the nucleic acid fragments of the invention; such novel vectors are also part of the invention. Details concerning the construction of these vectors of the invention will be discussed in context of transformed cells and microorganisms below. The vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector (and may be useful in DNA vaccination). Preferred cloning and expression vectors of the invention are capable of autonomous replication, thereby enabling high copy-numbers for the purposes of high-level expression or high-level replication for subsequent cloning.

The general outline of a vector of the invention comprises the following features in the 5'→3' direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma) of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator. When operating with expression vectors in producer strains or cell-lines it is for the purposes of genetic stability of the transformed cell preferred that the vector when introduced into a host cell is integrated in the host cell genome. In contrast, when working with vectors to be used for effecting in vivo expression in an animal {i.e. when using the vector in DNA vaccination) it is for security reasons preferred that the vector is not incapable of being integrated in the host cell genome; typically, naked DNA or non-integrating viral vectors are used, the choices of which are well-known to the person skilled in the art.

The vectors of the invention are used to transform host cells to produce the modified CRIPTO polypeptide of the invention. Such transformed cells, which are also part of the invention, can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors of the invention, or used for recombinant production of the modified polypeptides of the invention. Alternatively, the transformed cells can be suitable live vaccine strains wherein the nucleic acid fragment (one single or multiple copies) have been inserted so as to effect secretion or integration into the bacterial membrane or cell-wall of the modified CRIPTO.

Preferred transformed cells of the invention are microorganisms such as bacteria (such as the species Escherichia [e.g. E. coli], Bacillus [e.g. Bacillus subtilis], Salmonella, or Mycobacterium [preferably non-pathogenic, e.g. M. bovis BCG]), yeasts (such as

Saccharomyces cerevisiae), and protozoans. Alternatively, the transformed cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Most preferred are cells derived from a human being, cf. the discussion of cell lines and vectors below. Recent results have shown great promise in the use of a commercially available Drosophila melanogaster cell line (the Schneider 2 (S2) cell line and vector system available from Invitrogen) for the recombinant production of CRIPTO analogues of the invention, and therefore this expression system is particularly preferred, and therefore this type of system is also a preferred embodiment of the invention in general.

For the purposes of cloning and/or optimized expression it is preferred that the transformed cell is capable of replicating the nucleic acid fragment of the invention. Cells expressing the nucleic fragment are preferred useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the analogue or, in the case of non-pathogenic bacteria, as vaccine constituents in a live vaccine.

When producing the analogues of the invention by means of transformed cells, it is convenient, although far from essential, that the expression product is either exported out into the culture medium or carried on the surface of the transformed cell, since both of these options facilitate subsequent purification of the expression product.

When an effective producer cell has been identified it is preferred, on the basis thereof, to establish a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment encoding the modified CRIPTO. Preferably, this stable cell line secretes or carries the CRIPTO analogue of the invention, thereby facilitating purification thereof.

In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with the hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters that can be used by the prokaryotic microorganism for expression.

Those promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura ef al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979; EP-A-O 036 776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes may be expressed efficiently in E. coll from their own promoter sequences, precluding the need for addition of another promoter by artificial means.

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used, and here the promoter should be capable of driving expression. Saccharomyces cerevisiase, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman ef al., 1979; Tschemper ef al., 1980). This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells (commercially available as complete expression systems from La. Protein Sciences, 1000 Research Parkway, Meriden, CT 06450, U.S.A. and from Invitrogen), and MDCK cell lines. In the present invention, an especially preferred cell line the insect cell line S₂, available from Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands.

Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40) or cytomegalovirus (CMV). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers ef a/., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindlll site toward the BgII site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

LEGENDS TO FIGURES

Figure 1 : Structure model of CRIPTO made in the MODELER module of INSIGHT II software (Acclrys inc. San Diego) ²⁰. This model shows the structure in an Opened (A) and a closed conformation (B). The hydrophobic residues probably fold the molecule together.

Figure 2: Amino-acid sequence of the truncated CRIPTO molecule, hCRwt, used as template for generation of CRIPTO AutoVac™ molecules.

Figure 3 : Amino-acid sequences of hCRl CRIPTO AutoVac™ molecules.

Figure 4: Amino-acid sequences of hCR2 CRIPTO AutoVac™ molecules.

Figure 5: Amino-acid sequences of hCR3 CRIPTO AutoVac™ molecules.

Figure 6: Amino-acid sequences of hCR4 CRIPTO AutoVac™ molecules.

Figure 7: Amino-acid sequence of hEGFl CRIPTO peptide. PADRE sequence is underlined.

Figure 8: Amino-acid sequence of hCFCl CRIPTO peptide. PADRE sequence is underlined.

Figure 9: Amino-acid sequence of the synthetic full length wt CRIPTO protein received from GeneArt.

Figure 10a : Nucleotide sequence of the synthetic wt human CRIPTO gene. The hCRwt sequence used as template for the hCR constructs is marked in bold.

Figure 10b: Nucleotide sequence encoding the PADRE epitope.

Figure 10c: Nucleotide sequence encoding the HisTag.

Figure 11 : the hCRwt-p2Zop2f vector used as template for all hCR AutoVac™ constructs.

Figure 12: Schematic principle of SOE PCR. Principle of the polymerase chain reaction (PCR) based "gene Splicing by Overlap Extension" (SOE) method used in generation of the and hCR AutoVac™ constructs: Fragments from the genes that are to be recombined are generated in separate PCR reactions (Reaction 1 and 2). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed (Reaction 3), the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced¹ together. Addition of 5' and 3' oligo primers from Reaction 1 & 2, respectively, allows an exponential amplification of the spliced product. In the generation of the AutoVac™ constructs the splicing primers did not only provide the necessary complementary sequences but simultaneously introduced the PADRE T cell epitope.

Figure 13: CRIPTO DNA Vaccine constructs - including the full length CRIPTO sequence and the GPI anchor, inserted into the DNA vaccine vector, pCI.

EXAMPLES

EXAMPLE 1

Strategy for the molecular design of CRIPTO protein variants

Wild-type human CRIPTO template -hCRwt

Sequence alignments showed that areas of CRIPTO have high homology to EGF domains (Cys52-Arg81) and CFC domains (Cys85-Aspl20) and since both domains have three conserved disulfide bonds, these two domains might consist of the most conserved regions in human CRIPTO. Furthermore the CRIPTO sequence contains a signal peptide and a GPI linker for membrane attachment. As the signal sequence should be selected for the desired expression system, the original signal peptide is removed. As the GPI-linker could reduce the solubility of the protein or anchor the protein to cell membranes it is deleted in the truncated human CRIPTO sequence.

This truncated human wt CRIPTO molecule, named hCRwt, was used as template for construction of CRIPTO AutoVac™ molecules. The truncated human CRIPTO sequence consists of 140 residues starting at Leul (Leu31 in the cloned sequence¹⁹) and ending at Alal40 (Alal70 in the cloned sequence¹⁹) in the middle of the GPI-anchor sequence (Figure 2). Commercial recombinant human CRIPTO provided by R&D Systems, contains two N- terminals, Leu31 and Ser63 according to the product specification (Leul and Ser33 in hCRwt sequence). Therefore, if a site is exposed for degradation, point mutations can be made for inhibiting proteolysis.

Positions of expected disulfide bridges in the truncated human CRIPTO molecule are:

Cys52-Cys59; Cys53-Cys65; Cys67-Cys76; Cys85-CyslO3; Cys98-Cysll9 and CyslOl- CysllO.

CRIPTO AutoVac™ molecules

Ideal regions for inserting a foreign T cell epitope in human CRIPTO are localized in areas less conserved than the EGF and CFC domains. The chosen regions have mostly random coiled structures, where one might have freedom to insert a foreign T cell epitope. PADRE was chosen as foreign T cell epitope because of its small size.

Four regions in hCRwt have been selected for insertion of PADRE and so far, 12 CRIPTO AutoVac™ molecules have been designed.

hCRl AutoVac™ molecules: Sequence alignments show that the long sequence Leul-Pro39 (in hCRwt sequence) is unique to human CRIPTO (hCR). According to secondary structure prediction analysis this region consists of random coiled structure. Deletion of this region has been reported as not affecting the expression of the truncated molecule ²¹. Four CRIPTO AutoVac™ variants were constructed with PADRE inserted in this region and named hCRl.l, hCRl.2, hCRl.3, and hCRl.4 (Figure 3).

hCR2 AutoVac™ molecules: The short Met40-Thr51 amino-acid sequence has an unknown function. With PADRE insertion in this region, deletion of region hCRl can be obtained afterwards, as CRIPTO has been expressed without the hCRl region. Two CRIPTO AutoVac™ variants were constructed with PADRE inserted in this region and named hCR2.1 and hCR2.2 (Figure 4).

hCR3 AutoVac™ molecules: The Lys82-Asn84 amino-acid sequence constitutes a linker domain between the two most conserved regions of CRIPTO, i.e., the EGF and CFC domains. Three CRIPTO AutoVac™ variants were constructed with PADRE inserted in this region and named hCR3.1, hCR3.2, and hCR3.3 (Figure 5). hCR4 AutoVac™ molecules: The Glyl21-Alal40 amino-acid sequence constitutes the C- terminal region of CRIPTO, for which no function has been described. Three CRIPTO AutoVac™ variants were constructed with PADRE inserted in this region and named hCR4.1, hCR4.2, and hCR4.3 (Figure 6).

Single domain approach with synthetic peptides or recombinant proteins

It was previously reported that a synthetic EGF-like domain of human CRIPTO was functional after refolding¹⁹. Synthetic peptides or recombinant proteins -mapping to the EGF-like and CFC domains of CRIPTO and incorporating PADRE in their sequences- constitute the single domain CRIPTO AutoVac™ molecules. hEGFl and hCFCl molecules are examples of such single domain CRIPTO AutoVac™ molecules where PADRE is inserted in the C-terminal end of the peptides (Figures 7 and 8).

Removing degradation site by point mutation

In commercial available CRIPTO and Pharmexa-produced material two N-terminals are identified : Leul and Ser33 (numbered according to hCRwt sequence). Hydrophilic calculations for the area around Ser33 show that this region belongs to the most hydrophilic area of the molecule and therefore this region could be very accessible to proteolysis attach. One point mutation was made to possibly reduce this potential degradation site: Arg32Val. By replacement of Arg with VaI in position 32 the hydrophilicity is normalized and thereby the area is less accessible to proteolytic attach. The 50 residues long N-terminal part of human CRIPTO has an unknown function and it has only homology with CRIPTO related proteins.

New N-terminal Truncations of hCRwt template

It was decided to make new N-terminal truncations in hCRwt to increase the possibilities of producing a more homogeneous product with one single N-terminal. His-tagged versions of these constructs were made as well. Twelve new truncations are shown below in Table 1 hCRwt- T2 Arg30-Pro- ^■Arg- ^■Ser- ^■Ser- GIn- ^■Arg- ^■Val - ^■Pro hCRwt- T3 Pro31 -Arg- ^■Ser- ^■Ser- ^■Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro hCRwt- T4 Arg32 -Ser- ^■Ser- ^■Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro- ^■Met hCRwt- T5 Ser33-Ser- ^■Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro- ^■Met- ^■Gl y hCRwt- T 6 Ser34 -Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro- ^■Met- ^■Gly- ^■ He hCRwt- T7 Gln35-Arg- ^■Val - ^■Pro- ^■Pro- ^■Met- ^■Gly- ^■ I le- ^■Gin hCRwt- HIS-T2 Arg30- ^■Pro- ^■Arg- ^■Ser- ^■Ser- ^■Gln- ^■Arg- ^■Val- Pro hCRwt- HIS-T3 Pro31 - ^■Arg- ^■Ser- ^■Ser- ^■Gln- ^■Arg- ^■Val - ^■Pro- Pro hCRwt- HIS-T4 Arg32 - ^■Ser- ^■Ser- ^■Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro-Met hCRwt- HIS-T5 Ser33- ^■Ser- ^■Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro- ^■Met-Gly hCRwt- HIS-T 6 Ser34 - ^■Gln- ^■Arg- ^■Val - ^■Pro- ^■Pro- ^■Met- ^■Gl y- I le hCRwt- HIS-T7 Gln35- ^■Arg- ^■Val - ^■Pro- ^■Pro- ^■Met- ^■Gly- ^■ I le-Gln

Table 1 : 12 CRIPTO Analogues with N-terminal deletions

Introducing a Furin cleavage site by mutation

The introduction of a Furin cleavage site at Ser33 by mutations may be achieved by the following substitutions, Pro31S+K and Pro31Lys.

Choice of Tμ-epitope

At least three T_H-epitope containing peptides may be used for the CRIPTO variant design; these T_H-epitope containing peptides can be used alone or in combination within one CRIPTO variant: the tetanus toxoid epitope P2 (SEQ ID NO: 26AMEND), the tetanus toxoid epitope P30 (SEQ ID NO: 27) or a synthetic epitope of the PanDr family (e.g. SEQ ID NO: 21).

Choice of a CRIPTO isoform (Template)

The template for CRIPTO variant design is preferably CRIPTO hCRwt (SEQ ID No. 1), but the present design strategy is applicable to all naturally occurring CRIPTO isoforms, including SEQ ID No 16, and isoforms produced by alternative splicing and/or by proteolytic cleavage, as well as to the recombinantly expressed forms and to any truncated form that can, for example be produced by protease cleavage in vitro. Choice of insertion/substitution sites

Certain areas of native CRIPTO are believed to be superiorly suited for performing modifications for design of immunogenic variants of CRIPTO. It is for instance predicted that modifications within at least the following regions, Leul-Pro39, Met40-Thr51, Lys82-Asn84, Glyl21- Alal40 are considered most likely to produce the desired constructs and vaccination results. The main consideration for choosing these areas is the preservation in the variant of the tertiary structure of the CRIPTO protein.

Insertion/substitution within the N-terminus

Herein, the amino acid sequence SEQ ID NO: 29 is defined as the N-terminal region. This N- terminal region is selected as a primary target for T_H-epitope insertion/substitution since this region is poorly conserved within the CRYPTO family and it is structurally highly flexible showing no defined secondary structure elements. It is therefore a preferred site for insertion of foreign T-cell epitopes.

EXAMPLE 2

MOLECULAR CONSTRUCTION OF CRIPTO AUTOVAC™ MOLECULES

Design of the synthetic human CRIPTO DNA template

All human CRIPTO AutoVac™ molecules have been generated from a synthetic human CRIPTO DNA template designed at Pharmexa A/S in cooperation with GeneArt GMBH, (Regensburg, Germany) using the GeneOptimizer software program. The synthetic DNA sequence encoding the full length human CRIPTO polypeptide is a modified version of the cloned sequence described for human CRIPTO (accession P13385), as it has been codon- optimized for insect cells and CHO cells. The amino-acid sequence encoded by the synthetic wild type human CRIPTO is depicted in Figure 9 and the DNA sequence can be seen in Fig 10a. The human CRIPTO DNA sequence from base pair (bp) 51 to bp 510 was used to construct the hCRwt DNA template, which served as template for the generation of human CRIPTO AutoVac™ constructs.

Cloning of the synthetic hCRwt DNA template into the p2Zop2f expression vector

The synthetic human CRIPTO construct was delivered as a cloned and sequence-verified product in a pCR Script Amp vector backbone (Stratagene). Subcloning of the hCRwt coding sequence (CDS) from the pCR Script vector into the p2Zop2f expression vector (Research Coroporation Technologies) was done by polymerase chain reaction (PCR), by adding a Notl site immediately after the stop codon, and a sequence encoding a part of the Bip signal sequence (from p2Zop2f) upstream from and in-frame with the hCRwt gene with oligonucleotides (oligos) 2511+2507 (Example 3). This process omits the CRIPTO signal sequence, and prepares to replace it with the Bip signal sequence. The PCR product was isolated by agarose gel electrophoresis, purified (Example 8) and cloned into the pCR2.1- TOPO vector (Invitrogen) (Example 4). The resulting construct, hCRwt-pCR2.1-TOPO, was transformed into TOPlO cells (table 4) by electroporation (Example 10), and grown over night (ON) at 37°C, 220 rpm in 5 ml LB+ Kanamycin. The plasmid DNA was recovered (Example 11) and used as template to generate a PCR fragment containing the hCRwt gene with the part of the Bip sequence and a downstream part of the pCR2.1-TOPO vector (Example 3), using oligos 2507+1641.

Another PCR product was generated using a p2Zop2f vector with the Bip leader sequence as template, to generate a PCR fragment containing a large upstream region of p2Zop2f, and the Bip leader sequence after the promoter (Example 3). The two above mentioned PCR fragments were assembled using SOE PCR (Example 5 + Table T), the PCR fragment was gel- purified (Example 8), and the Bip leader sequence in frame with the hCRwt CDS was cloned as a Xhol/Notl fragment (Example 6) gel-purified (Example 8) and ligated (Example 9) with a p2Zop2f vector that had been digested with Xhol and Notl and dephosphorylated (Example 7). The ligation product was transformed into electrocompetent DHlOB cells (Example 10), plated out on LB plates with 30 mg/L Zeocin, and incubated at 37°C ON. Plasmid DNA was prepared (Example 11) from selected clones, sequenced and a correct clone was isolated and named hCRwt-p2Zop2f (Figure 11).

hCRwt-p2Zop2f was used as template for the construction of the hCR AutoVac™ constructs.

Molecular construction of single domain hCR-pET28b+ molecules

All single domain constructs are made from the synthetic human CRIPTO DNA template mentioned in section 6.1 using PCR and inserted in the relevant vectors.

Molecular construction of hCR-p2Zop2f AutoVac™ molecules

All human CRIPTO AutoVac™ molecules were constructed using SOE PCR (Example 3) using the primer and template combinations described in table 1. SOE PCR was used to insert the PADRE CDS (Figure 10b), in frame with the CRIPTO CDS, to generate genes encoding the human CRIPTO AutoVac™ proteins. Construction of hCRwt-HIS-p2Zop2f

To construct hCRwt-HIS-p2Zop2f, we have added a His-tag CDS (Figure 10c) in frame between the Bip signal CDS and the hCRwt CDS. This was done with SOE PCR (Example 3), with oligos 2557+2558.

Construction of truncated and degradation-optimized CRIPTO constructs

All truncated and cleavage-site mutated constructs are made from the hCRwt-p2Zop2f constructs using SOE-PCR and inserted in the p2Zop2f vector.

Table 2: Primer combinations and templates for the construction of hCR constructs.

Table 3: Oligonucleotide sequences

1967 CTCATGCGCGTGACCGGAC

Table 4 E.coli Strains used for cloning

EXAMPLE 3 : PCR cloning of the CRIPTO CDS

The following reaction mixture was added to each 0.2ml eppendorf tubes:

• 0.5μl template DNA

• 2.5μl (lOμM) oligo 2511

• 2.5μl (lOμM) oligo 2507

• 5μl dNTP (4 x 2.5mM)

• 5μl μl 1OX High Fidelity Expand buffer (Roche)

• 0.75μl 3.5U/μl High Fidelity Expand DNA polymerase (Roche)

• 33.75 μl H₂O

The samples were heated for 2 min. at 94°C in a T3 Thermo cycler (Biometra), then PCR was run for 20 touchdown cycles (94°C 15 sec; 60⁰C 30 sec; 72°C 1 min.) where the annealing temperature drops with 0.5⁰C in each cycle from the initial 60⁰C to 50⁰C in the last of the 20 cycles. The run was completed by a further 10 cycles (94°C 15 sec; 50⁰C 30 sec; 72°C 2 min. 10 sec) and 72°C 10 min, 4°C ∞.

EXAMPLE 4: Cloning in the pCR2.1-TOPO vector

The gel purified fragment was Taq treated to introduce A-overhangs, (15 min at 72°C with BioTaq polymerase, Taq-buffer and dNTP) and inserted into pCR2.1-TOPO vector from Invitrogen using the following conditions: lμl vector (pCR2.1-TOPO) lμl salt solution (Invitrogen) Diluted 4X 3μl purified PCR fragment incubated on ice 5 min, and electroporated into TOP-IO cells (Example 10).

EXAMPLE 5: Splicing by Overlap Extension (SOE by PCR)

SOE by PCR as used in the present work is based on the method described by Horton et al. ²⁴. The principle of the method is illustrated in Figure 12.

The combinations of oligonucleotide primers and templates are described in Table 1 and the exact oligonucleotide DNA sequences in Table 2. The oligonucleotide pairs summarized in the Oligos column in Table 1 reflect the different sub-reactions in the SOE method as described in Figure 12; First primer-pair for each construct represents reaction 1, the second primer-pair represents reaction 2 and the third primer-pair represents the respective 5' and 3' Reaction 1 & 2 primers added for final exponential amplification in Reaction 3.

The following reaction mixture added to each 0.2ml eppendorf tube:

• 0.5μl template DNA

• 2.5μl (lOμM) oligo I

• 2.5μl (lOμM) oligo II

• 5μl dNTP (4 x 2.5mM) • 5μl μl 1OX High Fidelity Expand buffer (Roche)

• 0.75μl 3.5U/μl High Fidelity Expand DNA polymerase (Roche)

• 33.75 μl H₂O

The samples were heated for 2 min. at 94°C in a T3 Thermo cycler (Biometra), then PCR was run for 20 touchdown cycles (94°C 15 sec; 60⁰C 30 sec; 72°C 1 min.) where the annealing temperature drops with 0.5⁰C in each cycle from the initial 60⁰C to 50⁰C in the last of the 20 cycles. The run was completed by 10 cycles as (94°C 15 sec; 50°C 30 sec; 72°C 2 min. 10 sec) and 72°C 10 min, 4°C ∞.

The product of Reaction 3 is purified via an agarose gel (Example 8), digested with Xhol and Notl restriction enzymes (Example 6) and purified again (Example 8). The digested product is subsequently ligated into the p2Zop2f vector and cloned in E.coli by traditional means (Example 7,9, and 10). EXAMPLE 6: Restriction enzyme digest

All restriction enzyme digestions were performed using the enzymes, buffers and (when recommended by the manufacturer), bovine serum albumin (BSA) - all supplied by New England Biolabs^βlnc (NEB). A standard reaction was typically performed in a total volume of 20μl but this was sometimes adjusted as required. Digestions with two different restriction enzymes in one reaction have been performed in the buffer recommended by NEB.

Xhol/Notl double digest of purified DNA product:

The following were mixed in a 1.5ml eppendorf tube

15μl DNA 2μl Notl enzyme (lO.OOOU/ml) lμl Xhol enzyme (20.000U/ml)

3μl 1OX NEB buffer 3

3μl 1OX BSA

6μl H₂O

The reaction was incubated at 37°C for 1.5 hour. The digested DNA was then ready for size separation and purification e.g. by agarose gel electrophoresis (Example 8)

EXAMPLE 7: Dephosphorylation of DNA 5' termini by Shrimp alkaline phosphatase (SAP) treatment.

Dephosphorylation of DNA 5' termini was used on the p2Zop2f expression vector and its derivates following digestion with restriction enzymes relevant for cloning of the hCR AutoVac™ inserts. The SAP treatment was performed in order to reduce the cloning-background of empty re-ligated vector.

SAP treatment of Notl/Xhol digested plasmid p2Zop2f:

P2Zop2f DNA was double-digested with Xhol and Notl restriction enzymes as described in Example 6. The digested vector was purified by agarose gel electrophoresis (Example 8). The purified vector was dephosphorylated with IX SAP buffer and 10 units of SAP (New England Biolabs) and incubated for 15 min at 37°C, and SAP was deactivated by incubation for 20 min at 65°C. The treated vector was then ready for ligation (Example 9) with an insert. EXAMPLE 8: Agarose gel electrophoresis and purification of DNA fragments

Agarose gel electrophoresis is a standard technique for visualization and/or separation of DNA fragments. The percentage of agarose in the gel is normally between 0.7% and 2% but should be adjusted to the size of the DNA molecules to be separated.

The agarose was mixed with IX TBE (0.9M Tris-borate, ImM EDTA). EtBr was added to the melted agarose to a final concentration of 0.05μg/ml. Electrophoresis was performed in a IX TBE running buffer with a voltage adapted the specific electrophoresis unit (around 100 volt, 500mAmp in a 12cm long electrophoresis unit). DNA samples were mixed with 6x Loading Dye Solution (MBI Fermentas) prior loading. A size marker was loaded next to the samples.

When properly separated by electrophoresis, the DNA fragment of interest was purified. All hCR related fragments were subsequently purified by use of a Qiaquick Gel Extraction kit (QIAGEN) using a microcentrifuge: After electrophoresis, the fragment was excised from the agarose gel and 3 volumes Buffer QG was added. The gel was dissolved by incubation at 50⁰C. The sample was applied to a QIAquick column prior centrifugation for 1 minute at > 10.000 x g. The column was washed in 0.75ml Buffer PE and centrifuged for 2 x 1 minute. The purified fragment was eluted by adding 50μl H₂O, and the DNA was collected by centrifugation for 1 minute.

EXAMPLE 9: DNA ligation

The following reaction describes the ligation of an hCR insert with p2Zop2f vector, and the method was applied for all ligations between the expression vector and hCR based AutoVac™ variant inserts.

The following were mixed in a 0.2ml eppendorf tube:

16μl hCR Xhol/Notl digested insert DNA (~30ng/ μl) lμl Xhol/Notl digested, SAP treated, p2Zop2f vector DNA (~0.1μg/μl) lμl T4 DNA ligase (400U/μl, NEB) 2μl 1OX T4 DNA ligase buffer (NEB)

Incubate for overnight in a T3 Thermo cycler (Biometra) in cycles as 30⁰C 30 sec; 10⁰C 30 sec; 30°C 30 sec. etc. EXAMPLE 10: Transformation of E.coli by electroporation

Electroporation was used for transformation of the cloning strains TOPlO (Invitrogen) and DH10B™ (Invitrogen).

Aliquots of the electrocompetent cells were thawed on ice, and the electroporation cuvettes were cooled on ice (0.1cm Bio Rad Cat. No. 165-2089).

25μl competent cells with mixed with each lμl ligation-mixture (as prepared in Example 9).

Electroporation was performed in a Bio Rad Micro Pulser, as described by the manufacturer (using a pulse at 1.8kV).

ImI of RT LB-medium was added and the electroporated cells were incubated with shaking at 37°C for 1 hour.

10 and lOOμl of the cell culture was then plated on LB-agar Zeocin or kanamycin plates and incubated at 37°C over night.

LB-medium and LB-agar zeocin/kanamycin plates are made as follows. LB-medium (IL) : 25g Lauria Broth (LB) 1000ml H₂O Autoclave.

LB-agar Zeocin/Kanamycin plates (10 x 25 ml) : 3.75g agar

250ml LB-medium

Autoclave.

Cool to 60⁰C, add kanamycin to a final concentration of 60μg/ml, or zeocin to a final concentration of 30 μg/ml, pour into petri-dishes.

EXAMPLE 11 : Plasmid DNA preparation

All plasmid DNA were prepared by the QIAGEN plasmid prep kit QIAprep® following the manufacturers recommended protocols. EXAMPLE 12: Recombinant expression of CRIPTO AutoVac™ molecules

Expression and production of recombinant CRIPTO molecules

Expression of human CRIPTO AutoVac™ molecules may be obtained in either insect or mammalian cells. As a first choice, expression of human CRIPTO proteins under the control of the OpIE2 promoter in Drosophila S2 cells was investigated. Drosophila S2 cells are known for fucosylating proteins. Mammalian cells could be considered as an alternative to Drosophila S2 cells.

EXAMPLE 13: Cultivation of Drosophila S2 cells

Drosophila S2 cells were cultivated in Excell420 (JRH Biosciences) in disposable shake flasks with vented cap or in disposable tissue culture flasks. The medium was fully replaced every three-five days and cells were diluted.

EXAMPLE 14: Transfection of Drosophila S2 cells

Drosophila S2 cells were transfected with the following vectors: p2312 and p2324-p2335 using Saint-18 in tissue culture flasks: 102 μl Saint-18 was mixed with 99 μl HBS. 2.25 μg DNA was mixed with HBS to a final volume of 225 μl and incubated for five minutes at room temperature. DNA and Saint-18 was mixed and added to 1.1E7 Drosophila S2 cells in 3.5 ml Excell420 with or without foetal bovine serum in a 25cm² tissue culture flask. The cell suspension was either 1) incubated for three-five days and a sample was taken in order to evaluate protein expression or 2) Zeocin was added to a final concentration of 1500 mg/l one day post-transfection in order to establish stable cell lines.

EXAMPLE 15: Establishment of stable cell lines.

Transfected Drosophila S2 cells were subjected to 1500 mg/l Zeocin one day post- transfection and cultivated in the presence of Zeocin until cells were growing stably and with a viability > 90%

EXAMPLE 16: Production of human CRIPTO AutoVac™ proteins in shake flasks

Stable cell lines were seeded in shake flasks at a cell density of 8E6 cells/ml in Excell 420 and cultivated for three-five days. In some cases, the medium was supplemented with 4 mM glutamine. The supernatant was harvested by centrifugation and saved at -2O⁰C. EXAMPLE 17: Production of human CRIPTO AutoVac™ proteins in bioreactor

Stable cell lines were expanded in shake flasks by centrifugation and re-suspension in fresh Excell420 to a final cell density of 8E6 cells/ml every three-four days. When a cell number of at least 15E9 cells was reached of each cell line, cells were pelleted and re-suspended in fresh Excell420 + 4 mM glutamine and inoculated in a bioreactor. The bioreactor consisted of a glass vessel equipped with pH electrode, DO electrode, temperature probe, level sensor and a cell retention device. Cells were cultivated for at least ten days in this perfusion system with a perfusion rate of 1 reactor volume per day. The perfusion harvest from each day was centrifuged, filtered and saved at -2O⁰C.

Detection of recombinant molecules by Coomassie gels

This semi-quantitative procedure may be used to estimate the concentrations of CRIPTO proteins in supernatant from S2 cells. The procedure is based on densitometry of CRIPTO protein bands in Coomassie stained SDS-PAGE gels of samples of unknown human CRIPTO concentration and consecutive determination of concentration using a human wt CRIPTO standard curve. Sample preparation: 20 μl sample is mixed with 20 μl 2x sample buffer.

Preparation of CRIPTO standards: 120 μl human wt CRIPTO with a concentration of e.g. 340 μg/ml is mixed with 40 μl 2x sample buffer. 100 μl of this solution is mixed 100 μl 2x sample buffer. 100 μl of this solution is mixed 100 μl 2x sample buffer and named Sl. 100 μl of this solution is mixed 100 μl 2x sample buffer and named S2. 100 μl of this solution is mixed 100 μl 2x sample buffer and named S3. A 12 well 12% Bis-Tris gel is loaded with 10 μl SeeBlue plus 2 Protein Marker, 20 μl Sl, 20 μl S2, 20 μl S3, and 20 μl of each of eight samples per gel. The gel is electrophoresed in Tris-Glycine buffer for ca. 90 minutes at 150 V. The gel is placed in fixing solution 15 minutes and washed 3x 5 minutes in MiIIi-Q water. The gel is stained in Blue Stain Reagent for one hour and rinsed and destained in MiIIi-Q water until the background is clear. The gel is scanned while wet using a Flat-bed scanner with scanning software, e.g. "HP ScanJet 7400C" with the "HP Precision Scan Pro 3.02" scanning software. After scanning, the image file is opened using the "Image Master" software. The software is used to generate a standard curve of the human wt CRIPTO bands (standards) and the concentration of the unknown samples will be calculated from this standard curve.

EXAMPLE 18: Purification and characterization of recombinant CRIPTO AutoVac™ molecules

Purification of wt human CRIPTO and recombinant CRIPTO AutoVac™ molecules was obtained in a three step purification procedure by: 1. Capto Q (IEX - anion exchange) : Efficient removal of Host Cell Proteins

2. SP (IEX - cation exchange) : Intermediate step - capture of CRIPTO

3. Superose 6 (SEC - size exclusion chromatography) : Removal of high molecular weight contaminants

Purification of the His-tagged wt human CRIPTO single domain molecules (EGF and CFC domain, receptively) involve IMAC (immobilized metal affinity chromatography) using either L-histidine or imidazole as eluent. If necessary, the HisTag can be cleaved off using the DAPase from Unizyme. Subsequent subtractive IMAC can be used to separate the cleaved off HisTag as well as the DAPase enzyme from the CRIPTO single domain molecules.

As E. coli may be used as expression host for the single domain molecules, refolding might be necessary in order to obtain properly folded domains which each contain three disulfide bonds. Refolding of the EGF and CFC domains as well as the folding of synthetic single domain CRIPTO peptides is carried out following a published method¹⁹.

The CRIPTO AutoVac™ molecules are analysed using the following methods:

-Amino acid analysis for determination of protein concentration, -N-terminal sequencing and Western blotting for CRIPTO identification, -purity determination using RP-HPLC and SDS-PAGE analysis, -secondary structure analysis using Circular Dichroism (CD), -size exclusion chromatography in combination with light scattering (SEC/LC) for analysis of aggregation

-temperature stability analysis using Differential Scanning Calorimetry (DSC), -peptide mapping for characterization of disulfide bonds and state of glycosylation.

EXAMPLE 19: Immunological selection of an AutoVac™ molecule for clinical development

Vaccination with CRIPTO AutoVac™ molecules and generation of antibodies

Immunizations: dose and schedule

Preclinical studies include hyper-immunization of mice in order to produce anti-CRIPTO antiserum for selection assays. In one example, groups of 50 mice are immunized with 20μg AutoVac™ molecule per dose, emulsified in an adjuvant such as Freund's adjuvant. Animals receive 4 immunizations at weeks 0, 2, 6, and 10. Blood samples are collected during the course of the experiment for evaluation of the humoral response. Two to three weeks after the last immunization animals are sacrificed and blood collected.

Antibody titer determination

A direct Enzyme-Linked ImmunoSorbant Assay (ELISA) is used to determine anti-CRIPTO antibody titers. MaxiSorb plates (Nunc, Denmark) are coated with 50 μl per well of a 4 μg/ml solution of human recombinant CRIPTO protein (RnDSystems, USA), diluted in a bicarbonate buffer (pH 9.6) for two hours at room temperature. Plates are washed three times in ELISA washing buffer containing 1 % Triton X-IOO. Plates are blocked with ELISA Blocking buffer (ELISA phosphate buffer saline (PBS) (pH 7.2) containing 1% bovine serum albumin (BSA) and 0.001% Phenol Red) for two hours at room temperature. After three additional washes, 50 μl/well of different dilutions of sera are added to the plates and incubated for 2 hours at room temperature. After three additional washes, secondary antibodies are added. If testing mouse sera, a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse antibody (p0260, DAKO, Denmark) diluted 1 : 1000 is added to all wells and incubated for 1 hour at room temperature. If using guinea pig sera, a horseradish peroxidase (HRP)-conjugated goat anti- guinea pig antibody (p0449, DAKO, Denmark) diluted 1 : 1000 is added to all wells and incubated for 1 hour at room temperature. Plates are washed five times, and 100 μl per well of TMB (plus) substrate is used for revelation. The reaction is stopped after 6 minutes by adding 100 μl/well of 2N H₂SO₄. Anti-CRIPTO antibody titers are determined relatively to a mouse monoclonal anti-CRIPTO antibody 50 μl/well, 1 μg/ml (MAB277, RnDSystems, USA).

Recognition of native CRIPTO molecules by antibodies raised by vaccination with CRIPTO AutoVac™

Polyclonal antibodies raised upon vaccination with CRIPTO AutoVac™ are tested for their ability to recognize native human CRIPTO molecules. An ELISA procedure can be used where wt CRIPTO molecules are coated in ELISA plates or alternatively, bound to coated anti- CRIPTO antibodies or recombinant Activin receptor IB. The generated polyclonal antibodies are added to the wells and detected with an HRP-conjugated antibody.

In another example, CRIPTO-expressing human tumor cell lines can be incubated with anti- CRIPTO polyclonal antibodies for detection of cell-surface CRIPTO molecules. After washing steps for elimination of unspecific binding, binding antibodies are detected with a fluorescent marker-conjugated antibody and visualized using a flow cytometry apparatus.

The binding affinities of anti-CRIPTO antibodies induced by different human CRIPTO variants can be measured using the Biacore equipment, where human native CRIPTO is bound to a sensor chip. Sera from vaccinated animals are tested for specific binding to the immobilized CRIPTO.

Yet another method to characterize the generated anti-CRIPTO antibodies is a functional ELISA where the anti-CRIPTO polyclonal antibodies are tested for their ability to inhibit the binding of wt CRIPTO molecules to coated recombinant Activin receptor IB, to coated recombinant Nodal molecules or to coated anti-CRIPTO antibodies.

Receptor binding ELISA assay

In the ELISA based inhibitory assay we set-up the experiment with CRIPTO and ActRIB/ALK- 4/Fc coated plates. In the first set-up plates are coated with human recombinant CRIPTO protein, and plates are blocked with 1% BSA in PBS. The binding of RIB/ALK-4/Fc chimera protein to CRIPTO is inhibited with anti-CRIPTO sera. CRIPTO bound RIB/ALK-4/Fc is detected with HRP labelled anti-human IgGl, which recognize the human Fc part (IgGl) of the RIB/ALK-4/Fc chimera protein. In the second assay we coat with 2 μg/ml RIB/ALK-4/Fc chimera protein to the microtitre plate and block the plate with PBS containing 1% BSA. Anti- CRIPTO sera from mice and guinea pigs are incubated with CRIPTO before being added to the RIB/ALK-4/Fc coated plates. Inhibition of CRIPTO binding is detected with biotinylated goat- anti-CRIPTO polyclonal antibody (BAF145). Assay is developed by addition of horseradish peroxidase-conjugated Streptavidin.

In vitro Competitive ELISA assay

A competitive ELISA procedure is used to measure recognition of native soluble CRIPTO proteins by polyclonal antibodies raised upon vaccination with CRIPTO AutoVac™. MaxiSorb (Nunc, Denmark) plates are coated with polyclonal anti-human CRIPTO wt protein, diluted in a bicarbonate buffer (pH 9.6) at room temperature. Plates are washed three times in ELISA washing buffer containing 1 % Triton X-IOO. Plates are blocked with 200 μl/well ELISA Blocking buffer (PBS (pH 7.2) containing 1% BSA and 0.001% Phenol Red for two hours at room temperature. A constant concentration of human CRIPTO wt protein is preincubated for 2 hours at room temperature with various dilutions of polyclonal sera from mice and guinea pigs. After blocking and three washes, 100 μl/well of different preincubations are added to the plates and incubated for 1 hour at room temperature. After three additional washes, 100 μl/well of a 0.5 μg/ml solution of biotinylated goat anti human CRIPTO (BAF145)

(RnDSystems, USA) are added to the plates and incubated for 1 hour at room temperature. After three additional washes, 100 μl/well of a horseradish peroxidase (HRP)-conjugated Streptavidin (Amersham) diluted 1 : 1000 are added to all wells and incubated for Vi hour at room temperature. Plates are washed five times, and 100 μl per well TMB+ substrate (Kem- en Tech, Denmark) are used for revelation. The reaction is stopped after 20 minutes by adding 100 μl/well of 2N H₂SO₄.

Antitumor activity of antibodies raised by vaccination with CRIPTO AutoVac™

In vitro tumor cell growth inhibition assay

Anti-CRIPTO polyclonal antibodies are tested for their ability to inhibit the in vitro growth of human tumor cell lines of different origins, such as MCF7 (breast cancer), LS174 (colon cancer), and DU145 (prostate cancer), as this was previously described for anti-CRIPTO monoclonal antibodies ¹⁷.

In vitro CRIPTO signaling assay

The generated anti-CRIPTO antibodies are tested for their ability to inhibit the CRIPTO- induced signal transduction in selected cell lines. Protein serine/threonine and tyrosine phosphorylation and Western blot analysis are performed with tumour cells i.e. NCCIT and Human umbilical vein endothelial cells (HUVEC) seeded in 60 mm diameter plates (1.5xlO⁵ cells/plate) and serum starved for 24 hours. Cells are stimulated with CRIPTO (200 or 400 ng/ml) +/- antibody at different concentrations at various times. The cells are lysed and protein samples (50 μg) are run on 10% SDS-PAGE and blotted to membranes. Blots are incubated with anti-phospho and total Smad-2, anti-phospho and total c-Src, anti-phospho and total MAPK, and anti-phospho and total Akt (serine 473) polyclonal antibodies over night at 4 degrees.

In vitro CRIPTO migration assay

The anti-CRIPTO antibodies are tested for their ability to inhibit tumor cell proliferation, migration and invasion. Cell migration and invasion assays are performed in fibronectin- coated Boyden chambers and Matrigel coated Boyden chambers. For both assays DMEM containing 5% fetal bovine serum is used in the lower Boyden chamber as the chemo- attractant. HUVEC cells are cultured in EBM-2 medium without serum and supplements, harvested by trypsination and resuspended in DMEM containing 5% bovine serum albumin at 4xlO⁵ cells per ml. 0.5 ml of this suspension is places in the upper chamber with or without the anti human CRIPTO antibody, CRIPTO (positive control) and inhibitors. The Boyden chambers were incubated overnight at 37 degrees celcius. Cells on the top side of the filter are removed and cells that had migrated and invaded the matrigel through the filter and attached to the bottom of the membrane are stained with crystal violet stain solution. Crystal violet stain solution is extracted with 10% acetic acid extraction buffer and transferred to wells of 96-multiwell plate and the absorbance is read at 595 nm.

Generation of suitable cell lines for CRIPTO in vitro assays

In order to develop a tumor cell line for the in vivo and in vitro studies expressing high levels of CRIPTO protein, human mammary epithelial cells, MCF-7 are transfected with a pCI vector construct containing the full length human CRIPTO sequence. Transfection of MCF-7 is performed with Effectene Transfection reagents (Qiagen). Stable clones are obtained by selective growth in G418 containg media.

In order to develop new in vitro assays for evaluation of produced anti-CRIPTO antisera, human embryonic kidney cells 293 (HEK 293) are transfected with pCI vector construct containing the full length human Activin receptor ALK-4. Transfection of 293 cells is performed with Effectene Transfection reagents (Qiagen). Stable clones are obtained by selective growth in G418 containg media.

In vivo Xenograft models

Anti-CRIPTO polyclonal antibodies are tested for their ability to inhibit the in vivo growth of human tumor cell lines of different origins, such as LS174 (colon cancer) and NCCIT (mediastinal mixed germ cell human testicular carcinoma). 2.5xlO⁵ cells in cell medium or in matrigel are implanted subcutaneously into SCID or Nude mice. Anti-CRIPTO antiserum is administered to the mice either before (prevention setting) or after (treatment setting) the tumor challenge, and tumor growth is recorded.

EXAMPLE 20: DNA Vaccines

DNA encoding hCRwt, hCR3.2 or full length CRIPTO including the GPI anchor are inserted into the DNA vaccine vector, pCI. The new DNA vaccine plasmids are named p2537, p2536, and p2498 respectively (SEQ ID 101, 102 and 103). Groups of C57BL/6 mice are immunized once intramuscularly with the naked DNA (100 microgram). Bulk T cell responses to hCRwt, hCR3.2, recombinant CRIPTO CFC domain as well as PADRE are analyzed in IFN-gamma ELISPOT assay. Preliminary results show that T cell responses are easily detected after immunization with each of the three vaccine constructs. Importantly, only C57BL/6 mice immunized with the hCR3.2 DNA vaccine show strong T cell response to PADRE when restimulated with the PADRE epitope. This result indicate that the DNA has been taken up by the APC, and that the protein has been produced, presented and has stimulated a specific T cell response. REFERENCES

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Claims

1. A method for in vivo down-regulation of CRIPTO activity in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of

a. at least one autologous CRIPTO protein or an autologous CRIPTO polypeptide or subsequence thereof which has been formulated so that immunization of the animal with the CRIPTO protein or CRIPTO polypeptide or subsequence thereof induces production of antibodies against the animal's autologous CRIPTO protein, and/or

b. at least one CRIPTO analogue, which comprises a CRIPTO polypeptide wherein is introduced at least one modification in the CRIPTO amino acid sequence which has as a result that immunization of the animal with the analogue induces production of antibodies against the animal's autologous CRIPTO protein.

2. The method according to claim 1, wherein is presented a CRIPTO analogue with at least one modification of the CRIPTO amino acid sequence.

3. The method according to claim 2, wherein the modification has as a result that a substantial fraction of CRIPTO B-cell epitopes is preserved.

4. The method according to any one of claims 1 to 3, wherein

■ at least one foreign T helper lymphocyte epitope (T_H epitope) is introduced, and/or

■ at least one first moiety is introduced which effects targeting of the modified molecule to an antigen presenting cell (APC) or a B-lymphocyte, and/or

■ at least one second moiety is introduced which stimulates the immune system, and/or ■ at least one third moiety is introduced which optimizes presentation of the modified CRIPTO polypeptide to the immune system.

5. The method according to any one of claims 2 to 4, wherein the modification has a result that at least one CRIPTO B-cell epitope is preserved.

6. The method according to claim 4 or 5, wherein the modification includes introduction as side groups, by covalent or non-covalent binding to suitable chemical groups in the CRIPTO polypeptide or a subsequence thereof, of the foreign T_H epitope and/or of the first and/or of the second and/or of the third moiety.

7. The method according to any one of claims 4 to 6, wherein the modification includes amino acid substitution and/or deletion and/or insertion and/or addition.

8. The method according to claim 7, wherein the modification results in the provision of a fusion polypeptide.

9. The method according to claim 7 or 8, wherein introduction of the amino acid substitution and/or deletion and/or insertion and/or addition results in a substantial preservation of the 3D structure of the CRIPTO polypeptide.

10. The method according to any one of claims 1 to 9, wherein the modification includes duplication of at least one CRIPTO B-cell epitope and/or introduction of a hapten.

11. The method according to any one of claims 4 to 10, wherein the foreign T-cell epitope is immunodominant in the animal.

12. The method according to any one of claims 4 to 11, wherein the foreign T-cell epitope is promiscuous.

13. The method according to claim 11 or 12, wherein at least one foreign T-cell epitope is selected from a natural promiscuous T-cell epitope and an artificial MHC-II binding peptide sequence.

14. The method according to claim 13, wherein the natural T-cell epitope is selected from a Tetanus toxoid epitope such as P2 or P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope, and wherein the artificial MHC-II binding peptide is a pan DR binding peptide.

15. The method according to any one of claims 4 to 14, wherein the first moiety is a substantially specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen, such as a hapten or a carbohydrate for which there is a receptor on the B-lymphocyte or the APC.

16. The method according to any one of claims 4 to 15, wherein the second moiety is selected from a cytokine, a hormone, and a heat-shock protein.

17. The method according to claim 16, wherein the cytokine is selected from, or is an effective part of, interferon γ (IFN-γ), Flt3L, interleukin 1 (IL-I), interleukin 2 (IL- T), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF), and the heat-shock protein is selected from, or is an effective part of any of, HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT).

18. The method according to any one of claims 4 to 17, wherein the third moiety is of lipid nature, such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group.

19. The method according to any one of the preceding claims, wherein the CRIPTO polypeptide is a human CRIPTO polypeptide, preferably SEQ ID NO: 16, or SEQ ID No 1, or allelic variant thereof.

20. The method according to claim 19, wherein the human CRIPTO polypeptide has been modified within the one or more of the following regions of SEQ ID No 1, or corresponding region of a CRIPTO polypeptide: Leul-Pro39; Met 40-Thr51, Lys82-

Asn84 and/or GIy 121-Alal40.

21. The method according to claim 20, wherein the human CRIPTO polypeptide has been modified by insertion into, deletion in, addition to, or substitution of any one of amino acids Leul-Pro39; Met 40-Thr51, Lys82-Asn84 and/or GIy 121-Alal40 of SEQ ID No 1, or corresponding position of a CRIPTO polypeptide.

22. The method according to any one of the preceding claims, wherein presentation to the immune system is effected by having at least two copies of the CRIPTO polypeptide, the subsequence thereof or the modified CRIPTO polypeptide covalently or non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants.

23. The method according to any one of the preceding claims, wherein the CRIPTO polypeptide, the subsequence thereof, or the modified CRIPTO polypeptide or CRIPTO analogue has been formulated with an adjuvant which facilitates breaking of autotolerance to autoantigens.

24. The method according to claim 23, wherein the adjuvant is selected from the group consisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (an ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ-inulin; and an encapsulating adjuvant.

25. The method according to any one of the preceding claims, wherein an effective amount of the CRIPTO polypeptide or the CRIPTO analogue is administered to the animal via a route selected from the parenteral route such as the intracutaneous, the subcutaneous, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the nasal route; the sublinqual route; the epidural route; the spinal route; the anal route; and the intracranial route.

26. The method according to claim 25, wherein the effective amount is between 0.5 μg and 2,000 μg of the CRIPTO polypeptide, the subsequence thereof or the analogue thereof.

27. The method according to claim 25 or 26, which includes at least one administration of the CRIPTO polypeptide or analogue per year, such as at least 2, at least 3, at least 4, at least 6, and at least 12 administrations per year.

28. The method according to any one of claims 25 to 27, wherein the CRIPTO polypeptide or analogue is contained in a virtual lymph node (VLN) device.

29. The method according to any one of claims 1 to 22, wherein presentation of modified CRIPTO to the immune system is effected by introducing nucleic acid(s) encoding the modified CRIPTO into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced.

30. The method according to claim 29, wherein the nucleic acid(s) introduced is/are selected from naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in chitin or chitosan, and DNA formulated with an adjuvant such as the adjuvants defined in claim 23 or 24.

31. The method according to claim 29 or 30, wherein the nucleic acids are administered intraarterially, intraveneously, or by the routes defined in claim 25.

32. The method according to any one of claims 29 to 31, wherein the nucleic acid(s) is/are contained in a VLN device.

33. The method according to any one of claims 29 to 32, which includes at least one administration of the nucleic acids per year, such as at least 2, at least 3, at least 4, at least 6, and at least 12 administrations per year.

34. The method according to any one of claims 1 to 33, wherein presentation to the immune system is effected by administering a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the CRIPTO polypeptide or analogue.

35. The method according to claim 34, wherein the virus is a non-virulent pox virus such as a vaccinia virus.

36. The method according to claim 35, wherein the microorganism is a bacterium, preferably a non pathogenic bacterium, such as a bacterium selected from the group consisting of a Mycobacterium spp. such as M.bovis BCG., Bacillus spp., Streptococcus spp., Escherichia spp., such as non-pathogenic strains of E. coli, Salmonella spp., Vibrio cholerae, and Shigella,

37. The method according to any one of claims 34 to 36, wherein the non-pathogenic microorganism or virus is administered one single time to the animal.

38. A method for the preparation of a CRIPTO vaccine capable of inducing in vivo down regulation of CRIPTO activity in an animal, including a human being, said method comprising either

a. Introducing at least one modification into at least one template molecule, wherein said template molecule is selected from the group comprising an autologous CRIPTO protein, a CRIPTO protein, CRIPTO polypeptide and a CRIPTO analogue,

and/or

b. combining at least one autologous CRIPTO protein, an autologous CRIPTO polypeptide or subsequence thereof, a CRIPTO analogue, or the modified template prepared in i), with at least one agent which effects targeting and/or presentation of the autologous CRIPTO protein or an autologous CRIPTO polypeptide to an antigen presenting cell (APC) or a B-lymphocyte and/or stimulates the immune system.

39. A method according to claim 38, wherein a substantial fraction of the CRIPTO B-cell epitopes are preserved in the CRIPTO vaccine.

40. A method according to claim 38, wherein at least one of the CRIPTO B-cell epitopes are preserved in the CRIPTO vaccine.

41. A method according to any one of claims 38 to 40, wherein said agent is selected from the group consisting of:

i. at least one first moiety which effects targeting of the CRIPTO vaccine to an antigen presenting cell (APC) or a B-lymphocyte, and/or ii. at least one second moiety is introduced which stimulates the immune system, and/or

iii. at least one third moiety is introduced which optimizes presentation of the modified CRIPTO polypeptide to the immune system,

42. The method according to claims 41, wherein the first moiety is a substantially specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen, such as a hapten or a carbohydrate for which there is a receptor on the B-lymphocyte or the APC.

43. The method according to claims 41 or 42, wherein the second moiety is selected from a cytokine, a hormone, and a heat-shock protein.

44. A method according to claim 43 wherein said second moiety is and/or an adjuvant which facilitates breaking of the autotolerence to autoantigens.

45. A method according to claim 44, wherein said adjuvant is selected from the group consisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ- inulin; and an encapsulating adjuvant.

46. The method according to any one of claims 43 - 45, wherein the cytokine is selected from, or is an effective part of, interferon γ (IFN-γ), Flt3L, interleukin 1 (IL-

1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL- 12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF), and the heat-shock protein is selected from, or is an effective part of any of, HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT).

47. The method according to any one of claims 41 to 46, wherein the third moiety is of lipid nature, such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group.

48. A method according to any one of claims 38 to 47, wherein said agent is a further copy of a CRIPTO protein, or a CRIPTO polypeptide, covalently or non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants.

49. A method according to any one of claim 38 to 48, wherein the vaccine is in the form of a nucleic acid fragment which encodes the modified CRIPTO in a form which is capable of entery into ithe animal's cells and thereby in vivo expression by the cells of the polynucleotide vaccine.

50. The method according to claim 49, wherein the nucleic acid fragment introduced is/are selected from naked DNA, DNA formulated with charged or uncharged lipids,

DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in chitin or chitosan, and DNA formulated with an adjuvant such as the adjuvants defined in any one of claims 43 - 46.

51. The method according to claim 50 wherein said nucleic acid fragment are within a non-pathogenic microorganism or virus.

52. The method according to claim 51, wherein the virus is a non-virulent pox virus such as a vaccinia virus.

53. The method according to claim 52, wherein the microorganism is a bacterium, preferably a non pathogenic bacterium, such as a bacterium selected from the group consisting of a Mycobacterium spp. such as M.bovis BCG., Bacillus spp., Streptococcus spp., Escherichia spp., such as non-pathogenic strains of E. coli, Salmonella spp., Vibrio cholerae, and Shigella,

54. A method according to any one of claims 38 to 53, wherein the template is selected from the group consisting of a SEQ ID No 1 and naturally occurring variants thereof, SEQ ID No 14 and naturally occurring variants thereof, SEQ ID No 15 and naturally occurring variants thereof, SEQ ID No 16 and naturally occurring variants thereof, an isolated EGF domain (such as SEQ ID No 22) or fragment thereof, and an isolated CFC domain (such as SEQ ID No 23) or fragment thereof.

55. A method according to any one of claims 38 to 54, wherein the modification includes amino acid substitution and/or deletion and/or insertion and/or addition.

56. A method according to claim 54 or 55, wherein the modification results in the provision of a fusion polypeptide.

57. The method according to claim 55 or 56, wherein introduction of the amino acid substitution and/or deletion and/or insertion and/or addition results in a substantial preservation of the 3D structure of the CRIPTO polypeptide.

58. The method according to any one of claims 38 to 57, wherein the modification includes duplication of at least one CRIPTO B-cell epitope and/or introduction of a hapten.

59. A method according to any one of claims 38 to 58, wherein said template is modified so as to introduce at least one foreign T cell helper epitope such as a T helper lymphocyte epitope /T_H epitope into said amino acid sequence, or is fused to said amino acid sequence, of said template.

60. The method according to claim 59, wherein the foreign T-cell epitope is immunodominant in the animal.

61. The method according to any one of claims 58 - 60, wherein the foreign T-cell epitope is promiscuous.

62. The method according to claim 61, wherein the at least one foreign T-cell epitope is selected from a natural promiscuous T-cell epitope and an artificial MHC-II binding peptide sequence.

63. The method according to any one of claims 59 to 62, wherein the foreign T-cell epitope is selected from a Tetanus toxoid epitope such as P2 or P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope, and an artificial MHC-II binding peptide such as a pan DR binding peptide.

64. A method according to any one of claims 59 to 63, wherein said at least one foreign T helper lymphocyte epitope /T_H epitope is inserted within the template sequence within one or more of the following regions of SEQ ID No 1, or equivalent position in an alternative CRIPTO template: Leul-Pro 39, Met40-51, Lys82-Asn84, and/or Glyl21-Alal40.

65. A method according to any one of claims 59 to 64, wherein the at least one foreign T helper lymphocyte epitope /T_H epitope is fused to the C and or N terminal of the template sequence.

66. A method according to any one of claims 38 to 65, wherein said template, CRIPTO protein, CRIPTO polypeptide and/or CRIPTO analogue comprises a mutation which has one or more mutations which improve in vivo stability.

67. A method according to any one of claims 38 to 66, wherein said template, CRIPTO protein, CRIPTO polypeptide and/or CRIPTO analogue comprises an N terminal truncation.

68. A method according to any one of claims 38 to 67, wherein said template, CRIPTO protein, CRIPTO polypeptide and/or CRIPTO analogue comprises a C terminal truncation.

69. A method according to any one of claims 38 to 68, wherein said template, or modified template prepared therefrom comprises a purification tag, such as a His tag.

70. A method according to any one of claims 38 to 69, wherein said CRIPTO vaccine comprises at least one of the cysteine bridges found in the autologous CRIPTO polypeptide SEQ ID No 16, such as at least 2, 3, 4, 5 or 6 of the cysteine bridges found in the autologous CRIPTO polypeptide SEQ ID No 16.

71. A CRIPTO vaccine prepared according to the method according to any one of claims 38 to 70.

72. A CRIPTO analogue which is derived from an animal CRIPTO polypeptide wherein is introduced a modification as defined in any one of claims 1-24 or 38 to 70.

73. The CRIPTO analogue according to claim 72, wherein said modification has as a result that immunization of the animal with the analogue induces production of antibodies against the CRIPTO polypeptide.

74. A vaccine preparation comprising the CRIPTO analogue according to claim 72 or 73.

75. An immunogenic composition comprising an immunogenically effective amount of a CRIPTO polypeptide autologous in an animal, said CRIPTO polypeptide being formulated together with an immunologically acceptable adjuvant so as to break the animal's autotolerance towards the CRIPTO polypeptide, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle.

76. An immunogenic composition comprising an immunogenically effective amount of a CRIPTO analogue according to claim 75, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant.

77. An immunogenic composition according to Claim 75 or 76, wherein the adjuvant is selected from the group consisting of the adjuvants according to any one of claims 43 - 46.

78. A method for in vivo down-regulation of CRIPTO activity in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of the CRIPTO vaccine according to any one of claims 71 - 74, or an immunogenic composition according to any one of claims 75 to 76.

79. A polypeptide consisting of one of the following amino acid sequences, SEQ ID No

1, SEQ ID No 22 and SEQ ID No 23.

80. A polypeptide comprising of one of the following amino acid sequences: SEQ ID No

2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15.

81. A nucleic acid fragment which encodes for one of the following polypeptides: SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15.

82. A nucleic acid fragment which encodes a CRIPTO vaccine according to claim 71 or a CRIPTO analogue according to claim 72.

83. A vector carrying the nucleic acid fragment according to claim 81 or 82

84. The vector according to claim 83 which is capable of autonomous replication.

85. The vector according to claim 83 or 84 which is selected from the group consisting of a plasmid, a phage, a cosmid, a mini-chromosome, and a virus.

86. The vector according to any one of claims 83 to 85, comprising, in the 5'→3' direction and in operable linkage, a promoter for driving expression of the nucleic acid fragment according to claim 81 or 82, optionally a nucleic acid sequence encoding a leader peptide enabling secretion of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment according to claim 81 or 82, and optionally a terminator.

87. The vector according to any one of claims 83 to 85 which, when introduced into a host cell, is integrated in the host cell genome.

88. The vector according to any one of claims 83 to 86 which, when introduced into a host cell, is not capable of being integrated in the host cell genome.

89. The vector according to any one of claims 83 to 88, wherein the promoter drives expression in a eukaryotic cell and/or in a prokaryotic cell.

90. A transformed cell carrying the vector of any one of claims 83-89.

91. The transformed cell according to claim 90 which is capable of replicating the nucleic acid fragment according to claim according to claim 81 or 82.

92. The transformed cell according to claim 91, which is a microorganism selected from a bacterium, a yeast, a protozoan, or a cell derived from a multicellular organism selected from a fungus, an insect cell such as an S₂ or an SF cell, a plant cell, and a mammalian cell.

93. The transformed cell according to claim 92 which is a bacterium of the genus Escherichia, Bacillus, Salmonella, or Mycobacterium.

94. The transformed cell according to claim 93, which is selected from the group consisting of an E. coli cell, and a non-pathogenic Mycobacterium cell such as M. bovis BCG.

95. The transformed cell according to any one of claims 90-94, which expresses the nucleic acid fragment according to claim 81 or 82.

96. The transformed cell according to claim 95, which secretes or carries on its surface, the CRIPTO analogue according to claim 74.

97. A method for treating and/or preventing and/or ameliorating diseases selected from the group consisting of malignant neoplasm, benign neoplasm, inflammatory diseases, and diabetes and diabetes related conditions, the method comprising down-regulation of CRIPTO according to any one claims 1 to 37 or claim 78.

98. A composition for inducing production of antibodies against CRIPTO, the composition comprising

a. a nucleic acid fragment according to claim 81 or 82 or a vector according to any one of claims 83 to 89, and

b. a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or adjuvant.

99. The composition according to claim 98, wherein the nucleic acid fragment is formulated according to any one of claims 30 to 32.

100. A stable cell line which carries the vector according to any one of claims 83 to 89 and which expresses the nucleic acid fragment according to claim 81 or 82, and which optionally secretes or carries the CRIPTO analogue according to claim 72 on its surface.

101. A method for the preparation of the cell according to any one of claims 90-96, the method comprising transforming a host cell with the nucleic acid fragment according to claim 81 or 82 or with the vector according to any one of claims 82 to 89.

102. Use of the CRIPTO vaccine and/or analogue according to any one of claims 71 - 74 in the manufacture of a pharmaceutical composition for use in the in vivo down- regulation of CRIPTO activity in an animal, including a human being.