WO2008040759A1 - Procédé destiné à réduire le nombre de cripto - Google Patents

Procédé destiné à réduire le nombre de cripto Download PDF

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WO2008040759A1
WO2008040759A1 PCT/EP2007/060502 EP2007060502W WO2008040759A1 WO 2008040759 A1 WO2008040759 A1 WO 2008040759A1 EP 2007060502 W EP2007060502 W EP 2007060502W WO 2008040759 A1 WO2008040759 A1 WO 2008040759A1
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Prior art keywords
cripto
seq
polypeptide
cell
epitope
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PCT/EP2007/060502
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English (en)
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Valéry RENARD
Dana Leach
Steen Klysner
Peter Birk Rasmussen
Finn Stausholm Nielsen
Tomas Bratt
Bjørn VOLDBORG
Florence Dal Degan
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Pharmexa A/S
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Publication of WO2008040759A1 publication Critical patent/WO2008040759A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins

Definitions

  • 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.
  • 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.
  • 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.
  • EGF epidermal growth factor
  • CFC Cryptic domain
  • 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.
  • CRIPTO mRNA 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.
  • CRIPTO Crohn's disease
  • 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 .
  • CRIPTO forms complex with activin and type II activin receptors, which seems to prevent the formation of activin/ activin receptors complexes 12 .
  • transfection of the CRIPTO gene into different cell lines expressing activin receptors resulted in the inhibition of activin signaling.
  • 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.
  • 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.
  • Glypican-1 is elevated in human breast cancer, whereas its expression is low in normal breast tissue 13 .
  • Glypican may act to promote the growth- promoting effect of CRIPTO in breast cancer cells.
  • mAbs monoclonal antibodies
  • 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.
  • 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 glycosylations 19 .
  • CRIPTO 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.
  • each of the EGF-like and CFC domains have a much conserved disulfide bond pattern with three disulfide bridges in each domain.
  • 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 20 .
  • the degree of standard structure elements like ⁇ -helixes and ⁇ -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).
  • active immunotherapy as a means of curing or alleviating disease has received growing attention over the last 2 decades.
  • 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).
  • 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.
  • 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.
  • CTLs cytotoxic T-lymphocytes
  • 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.
  • 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 reported 17 ' 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.
  • 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.
  • the anti-CRIPTO immune response wanes.
  • 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
  • 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.
  • 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
  • APC 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.
  • a CRIPTO vaccine such as CRIPTO analogues suitable for use as a CRIPTO vaccine, prepared according to the above method.
  • 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.
  • 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.
  • an immunogenic composition such as a CRIPTO vaccine
  • 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.
  • 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.
  • an immunogenic composition such as a CRIPTO vaccine
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • NCBI protein database 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.
  • 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.
  • 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.
  • the polypeptide may give rise to an immune response against CRIPTO of the animal in question.
  • 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.
  • 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 leas ⁇ 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).
  • 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.
  • 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.
  • 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 leas ⁇ 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
  • 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).
  • 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
  • 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. ).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • pharmaceutical 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.
  • 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.
  • CRIPTO has been "modified"
  • a chemical modification of the polypeptide which constitutes the backbone of CRIPTO 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.
  • 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.
  • 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” 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • substantially specific binding partner includes in one embodiment the term "specific binding partner”.
  • 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.
  • T H epitope foreign T helper lymphocyte epitope
  • APC antigen presenting cell
  • B-lymphocyte an antigen presenting cell
  • at least one second moiety is introduced which stimulates the immune system
  • at least one third moiety is introduced which optimizes presentation of the modified CRIPTO polypeptide to the immune system.
  • 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 ⁇ (IFN- ⁇ ), 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).
  • IFN- ⁇ interferon ⁇
  • Flt3L interleukin 1
  • IL-2 interleukin 2
  • IL-4 interleukin 4
  • IL-6 interleukin 6
  • IL-12 interleukin 12
  • IL-13 interleukin 13
  • IL-15
  • a preferred heat-shock protein is, or is an effective part of any of, HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT).
  • 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.
  • 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.
  • 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.
  • this embodiment allows for substitution or insertion of the protein, as long as the 3D structure of the protein is maintained.
  • 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.
  • Modified versions 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • T helper cell epitope 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.
  • 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.
  • the epitope can be any artificial T-cell epitope which is capable of binding a large proportion of MHC Class II molecules.
  • the pan DR epitope peptides PADRE
  • 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.
  • 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
  • an immunologically effective subsequence thereof is 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.
  • the introduction of a foreign T-cell epitope can be accomplished by introduction of at least one amino acid insertion, addition, deletion, or substitution.
  • 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.
  • APC antigen presenting cell
  • 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.
  • 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.
  • T H epitope foreign MHC Class II binding amino acid sequence
  • the question of immune dominance of a T H epitope depends on the animal species in question.
  • 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.
  • T H epitopes An important point is the issue of MHC restriction of T H epitopes.
  • 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.
  • the fraction of the animal population covered by a specific vaccine composition can be determined by means of the following formula:
  • ⁇ ⁇ 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 ⁇ lr ⁇ f ⁇ , and ⁇ 3 .
  • 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).
  • DP 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).
  • 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.
  • 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.
  • 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.
  • CRIPTO analogues such as immunogenic analogues
  • autologous CRIPTO polypeptides may be used.
  • 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 glycosylations 19 .
  • CRIPTO 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.
  • each of the EGF-like and CFC domains have a much conserved disulfide bond pattern with three disulfide bridges in each domain.
  • 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.
  • 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).
  • the CRIPTO analogue according to the invention retains the six disulphide bridges found in the human CRIPTO protein.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 AutoVacTM 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.
  • 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 AutoVacTM 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.
  • 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.
  • 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.
  • 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).
  • fragments of such single domains may also be used as long as the fragment comprises at least one B cell epitope.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 compations 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 compations 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.
  • 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.
  • a number of purification tag systems have been developed to facilitate and standardize purification of recombinant proteins 22 ' 23 .
  • 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.
  • IMAC immobilized metal affinity chromatography
  • 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 MKHQHQHQHQHQAP (SEQ ID No 98).
  • the HisTag may be fused to the C-terminus of the CRIPTO molecule.
  • 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.
  • DAPase dipeptidyl amino-peptidase I
  • 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.
  • 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.
  • two or more CRIPTO sequences such as CRIPTO proteins, polypeptides or analogues may be fused or otherwise joined together.
  • 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.
  • spacer sequence such as a short glycine linker.
  • 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 may be performed between two CRIPTO analogues, which may be the same or different.
  • the formulation of the polypeptide follows the principles generally acknowledged in the art.
  • vaccines which contain peptide sequences as active ingredients are 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.
  • 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.
  • 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.
  • 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%.
  • cholera toxin is an interesting formulation partner (and also a possible conjugation partner).
  • 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.
  • 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
  • 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 ⁇ g to 2,000 ⁇ g (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 ⁇ g to 1,000 ⁇ g, preferably in the range from 1 ⁇ g to 500 ⁇ g and especially in the range from about 10 ⁇ g to 100 ⁇ g.
  • 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.
  • a polypeptide construct of the invention 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.
  • 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.
  • 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.
  • 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; ⁇ - inulin; and an encapsulating adjuvant.
  • 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
  • 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 101 0 C 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.
  • 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 101 0 C for 30 second to 2 minute periods respectively and
  • 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.
  • DDA dimethyldioctadecylammonium bromide
  • DNA and ⁇ -inulin are interesting candidates for an adjuvant as is DNA and ⁇ -inulin, but also Freund's complete and incomplete adjuvants as well as quillaja saponins such as QuilA and QS21 are interesting as is RIBI.
  • MPL monophosphoryl lipid A
  • C3 and C3d are preferred adjuvant
  • Provax® Biogen
  • Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants are preferred according to the invention.
  • 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.
  • the resulting particulate formulation 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.
  • the saponin constitutes 60-70% w/w
  • the cholesterol and phospholipid 10-15% w/w constitutes 60-70% w/w
  • the protein 10-15% w/w 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.
  • a relevant antigen such as an antigen of the present invention
  • the presentation of a relevant antigen can be enhanced by conjugating the antigen to antibodies (or antigen binding antibody fragments) against the Fc ⁇ receptors on monocytes/macrophages.
  • conjugates between antigen and anti-Fc ⁇ RI have been demonstrated to enhance immunogenicity for the purposes of vaccination.
  • 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.
  • a "virtual lymph node” VLN
  • the VLN (a thin tubular device) mimics the structure and function of a lymph node.
  • 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.
  • 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.
  • 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.
  • 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.
  • bio bio
  • 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.
  • 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.
  • 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
  • nucleic acid vaccines can suitably be administered intraveneously and intraarterially.
  • 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.
  • VLN 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.
  • 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.
  • 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.
  • the invention also relates to a composition for inducing production of antibodies against CRIPTO, the composition comprising
  • nucleic acid fragment or a vector of the invention (cf. the discussion of nucleic acids and vectors below), and
  • nucleic acid is introduced in the form of a vector wherein expression is under control of a viral promoter.
  • vectors and DNA fragments according to the invention cf. the discussion below.
  • 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.
  • a third alternative for effecting presentation of the analogues of the invention to the immune system is the use of live vaccine technology.
  • 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.
  • 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.
  • 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.
  • live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination.
  • 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.
  • two distinct nucleotide fragments can be used as transforming agents.
  • 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.
  • 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.
  • Angiogenesis provides then an attractive therapeutic target for therapy of solid tumours and with a theoretically limited toxicity profile.
  • anti-CRIPTO antibody therapy demonstrated potent anti-tumor activity in different cancers including breast, pancreatic, colon, lung, and ovarian cancers.
  • compositions of the invention are provided.
  • the invention also pertains to compositions useful in exercising the method of the invention.
  • 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.
  • 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.
  • 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.
  • protein carriers such as KLH, diphtheria toxoid, tetanus toxoid, and BSA
  • non-proteinaceous molecules such as carbohydrate polymers
  • nucleic acid fragments encoding the analogues are important chemical products (as are their complementary sequences).
  • 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.
  • 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.
  • vectors to be used for effecting in vivo expression in an animal ⁇ i.e. when using the vector in DNA vaccination
  • the vector 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.
  • 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
  • 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.
  • S2 Drosophila melanogaster cell line
  • Invitrogen the Schneider 2 (S2) cell line and vector system available from Invitrogen
  • 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.
  • this stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment encoding the modified CRIPTO.
  • this stable cell line secretes or carries the CRIPTO analogue of the invention, thereby facilitating purification thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • 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).
  • 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.
  • an especially preferred cell line the insect cell line S 2 , 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.
  • control functions on the expression vectors are often provided by viral material.
  • promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40) or cytomegalovirus (CMV).
  • SV40 Simian Virus 40
  • CMV cytomegalovirus
  • 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.
  • 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.
  • 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.
  • 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 AutoVacTM molecules.
  • Figure 3 Amino-acid sequences of hCRl CRIPTO AutoVacTM molecules.
  • Figure 4 Amino-acid sequences of hCR2 CRIPTO AutoVacTM molecules.
  • Figure 5 Amino-acid sequences of hCR3 CRIPTO AutoVacTM molecules.
  • Figure 6 Amino-acid sequences of hCR4 CRIPTO AutoVacTM 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 AutoVacTM 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 AutoVacTM 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 'spliced 1 together.
  • PCR polymerase chain reaction
  • SOE Gene Splicing by Overlap Extension
  • FIG. 13 CRIPTO DNA Vaccine constructs - including the full length CRIPTO sequence and the GPI anchor, inserted into the DNA vaccine vector, pCI.
  • CRIPTO sequence contains a signal peptide and a GPI linker for membrane attachment.
  • the signal sequence should be selected for the desired expression system, the original signal peptide is removed.
  • 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 was used as template for construction of CRIPTO AutoVacTM molecules.
  • the truncated human CRIPTO sequence consists of 140 residues starting at Leul (Leu31 in the cloned sequence 19 ) and ending at Alal40 (Alal70 in the cloned sequence 19 ) 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.
  • 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.
  • hCRl AutoVacTM 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 .
  • CRIPTO AutoVacTM variants were constructed with PADRE inserted in this region and named hCRl.l, hCRl.2, hCRl.3, and hCRl.4 ( Figure 3).
  • hCR2 AutoVacTM 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 AutoVacTM variants were constructed with PADRE inserted in this region and named hCR2.1 and hCR2.2 ( Figure 4).
  • hCR3 AutoVacTM 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 AutoVacTM variants were constructed with PADRE inserted in this region and named hCR3.1, hCR3.2, and hCR3.3 ( Figure 5).
  • hCR4 AutoVacTM molecules The Glyl21-Alal40 amino-acid sequence constitutes the C- terminal region of CRIPTO, for which no function has been described. Three CRIPTO AutoVacTM variants were constructed with PADRE inserted in this region and named hCR4.1, hCR4.2, and hCR4.3 ( Figure 6).
  • Furin cleavage site at Ser33 by mutations may be achieved by the following substitutions, Pro31S+K and Pro31Lys.
  • 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).
  • 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.
  • 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.
  • 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.
  • All human CRIPTO AutoVacTM 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 AutoVacTM constructs.
  • 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 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.
  • 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).
  • 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 AutoVacTM 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.
  • 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; 60 0 C 30 sec; 72°C 1 min.) where the annealing temperature drops with 0.5 0 C in each cycle from the initial 60 0 C to 50 0 C in the last of the 20 cycles.
  • the run was completed by a further 10 cycles (94°C 15 sec; 50 0 C 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: l ⁇ l vector (pCR2.1-TOPO) l ⁇ l salt solution (Invitrogen) Diluted 4X 3 ⁇ l purified PCR fragment incubated on ice 5 min, and electroporated into TOP-IO cells (Example 10).
  • 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 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; 60 0 C 30 sec; 72°C 1 min.) where the annealing temperature drops with 0.5 0 C in each cycle from the initial 60 0 C to 50 0 C 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
  • EXAMPLE 7 Dephosphorylation of DNA 5' termini by Shrimp alkaline phosphatase (SAP) treatment.
  • 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 0.05 ⁇ g/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.
  • 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 50 0 C. 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 50 ⁇ l H 2 O, and the DNA was collected by centrifugation for 1 minute.
  • QIAGEN Qiaquick Gel Extraction kit
  • Electroporation was used for transformation of the cloning strains TOPlO (Invitrogen) and DH10BTM (Invitrogen).
  • 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.
  • LB-medium and LB-agar zeocin/kanamycin plates are made as follows.
  • LB-medium (IL) 25g Lauria Broth (LB) 1000ml H 2 O Autoclave.
  • human CRIPTO AutoVacTM molecules may be obtained in either insect or mammalian cells.
  • 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.
  • 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.
  • Drosophila S2 cells were transfected with the following vectors: p2312 and p2324-p2335 using Saint-18 in tissue culture flasks: 102 ⁇ l Saint-18 was mixed with 99 ⁇ l HBS. 2.25 ⁇ g DNA was mixed with HBS to a final volume of 225 ⁇ l 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 25cm 2 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%
  • 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.
  • 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 -2O 0 C.
  • 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 ⁇ l sample is mixed with 20 ⁇ l 2x sample buffer.
  • CRIPTO standards 120 ⁇ l human wt CRIPTO with a concentration of e.g. 340 ⁇ g/ml is mixed with 40 ⁇ l 2x sample buffer. 100 ⁇ l of this solution is mixed 100 ⁇ l 2x sample buffer. 100 ⁇ l of this solution is mixed 100 ⁇ l 2x sample buffer and named Sl. 100 ⁇ l of this solution is mixed 100 ⁇ l 2x sample buffer and named S2. 100 ⁇ l of this solution is mixed 100 ⁇ l 2x sample buffer and named S3. A 12 well 12% Bis-Tris gel is loaded with 10 ⁇ l SeeBlue plus 2 Protein Marker, 20 ⁇ l Sl, 20 ⁇ l S2, 20 ⁇ l S3, and 20 ⁇ l 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.
  • His-tagged wt human CRIPTO single domain molecules EGF and CFC domain, receptively
  • IMAC immobilized metal affinity chromatography
  • 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.
  • 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 method 19 .
  • the CRIPTO AutoVacTM molecules are analysed using the following methods:
  • DSC Differential Scanning Calorimetry
  • mice Preclinical studies include hyper-immunization of mice in order to produce anti-CRIPTO antiserum for selection assays.
  • groups of 50 mice are immunized with 20 ⁇ g AutoVacTM 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.
  • ELISA Enzyme-Linked ImmunoSorbant Assay
  • ELISA Blocking buffer ELISA phosphate buffer saline (PBS) (pH 7.2) containing 1% bovine serum albumin (BSA) and 0.001% Phenol Red
  • PBS ELISA phosphate buffer saline
  • BSA bovine serum albumin
  • Phenol Red 50 ⁇ l/well of different dilutions of sera are added to the plates and incubated for 2 hours at room temperature.
  • secondary antibodies are added.
  • HRP horseradish peroxidase
  • p0260, DAKO, Denmark diluted 1 : 1000 is added to all wells and incubated for 1 hour at room temperature.
  • HRP horseradish peroxidase
  • a horseradish peroxidase (HRP)-conjugated goat anti- guinea pig antibody diluted 1 : 1000 is added to all wells and incubated for 1 hour at room temperature. Plates are washed five times, and 100 ⁇ l per well of TMB (plus) substrate is used for revelation. The reaction is stopped after 6 minutes by adding 100 ⁇ l/well of 2N H 2 SO 4 .
  • Anti-CRIPTO antibody titers are determined relatively to a mouse monoclonal anti-CRIPTO antibody 50 ⁇ l/well, 1 ⁇ g/ml (MAB277, RnDSystems, USA).
  • Polyclonal antibodies raised upon vaccination with CRIPTO AutoVacTM 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.
  • 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.
  • 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.
  • a competitive ELISA procedure is used to measure recognition of native soluble CRIPTO proteins by polyclonal antibodies raised upon vaccination with CRIPTO AutoVacTM.
  • 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 ⁇ l/well ELISA Blocking buffer (PBS (pH 7.2) containing 1% BSA and 0.001% Phenol Red for two hours at room temperature.
  • PBS pH 7.2
  • 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 ⁇ l/well of different preincubations are added to the plates and incubated for 1 hour at room temperature. After three additional washes, 100 ⁇ l/well of a 0.5 ⁇ g/ml solution of biotinylated goat anti human CRIPTO (BAF145)
  • HRP horseradish peroxidase
  • Amersham horseradish peroxidase
  • diluted 1 : 1000 are added to all wells and incubated for Vi hour at room temperature. Plates are washed five times, and 100 ⁇ l per well TMB+ substrate (Kem- en Tech, Denmark) are used for revelation. The reaction is stopped after 20 minutes by adding 100 ⁇ l/well of 2N H 2 SO 4 .
  • 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 .
  • 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 (1.5xlO 5 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 ⁇ g) 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.
  • 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.
  • 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 4xlO 5 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.
  • 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.
  • 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.
  • 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). 2.5xlO 5 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.
  • 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.
  • CRIPTO a member of the epidermal growth factor family, is over-expressed in human pancreatic cancer and chronic pancreatitis. Int.J. Cancer 56:668-674.
  • CRIPTO a novel gene of the epidermal growth factor gene family, leads to in vitro transformation of a normal mouse mammary epithelial cell line. Cancer Res. 51 : 1051- 1054. 7. Niemeyer,C.C, M.G.Persico, and E.D.Adamson. 1998. CRIPTO: roles in mammary cell growth, survival, differentiation and transformation. Cell Death. Differ. 5:440-449.
  • Epithelial mesenchymal transition is a characteristic of hyperplasias and tumors in mammary gland from M MTV-CRI PTOl transgenic mice. J. Cell Physiol 201 :266-276. 12. Gray,P.C, C.A.Harrison, and W.Vale. 2003. CRIPTO forms a complex with activin and type II activin receptors and can block activin signaling. Proc.Natl.Acad.Sci.U.S.A 100:5193-5198.
  • Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells.

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Abstract

La présente invention concerne des variantes immunogènes de CRIPTO qui sont utiles dans l'immunothérapie spécifique active contre des maladies caractérisées par une surexpression de CRIPTO. L'invention concerne également des procédés destinés au traitement de telles maladies (par exemple le cancer), ainsi que des outils divers en biologie moléculaire qui aident à fournir des variantes immunogènes.
PCT/EP2007/060502 2006-10-03 2007-10-03 Procédé destiné à réduire le nombre de cripto WO2008040759A1 (fr)

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WO2002022808A2 (fr) * 2000-09-18 2002-03-21 Biogen, Inc. Mutant cripto et utilisations de ce dernier
EP1502602A2 (fr) * 1998-10-05 2005-02-02 Pharmexa A/S Procedes de vaccination therapeutique
WO2005042575A2 (fr) * 2003-10-30 2005-05-12 Pharmexa A/S Procede de regulation negative du facteur de croissance endotheliale vasculaire
EP1632242A2 (fr) * 2000-02-21 2006-03-08 Pharmexa A/S Méthode nouvelle pour la régulation négative de l'amyloide

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EP1502602A2 (fr) * 1998-10-05 2005-02-02 Pharmexa A/S Procedes de vaccination therapeutique
EP1632242A2 (fr) * 2000-02-21 2006-03-08 Pharmexa A/S Méthode nouvelle pour la régulation négative de l'amyloide
WO2002016413A2 (fr) * 2000-08-24 2002-02-28 Glaxosmithkline Biologicals S.A. Vaccins
WO2002022808A2 (fr) * 2000-09-18 2002-03-21 Biogen, Inc. Mutant cripto et utilisations de ce dernier
WO2005042575A2 (fr) * 2003-10-30 2005-05-12 Pharmexa A/S Procede de regulation negative du facteur de croissance endotheliale vasculaire

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105474076A (zh) * 2013-08-30 2016-04-06 国际商业机器公司 状态可改变装置
US10102906B2 (en) 2013-08-30 2018-10-16 International Business Machines Corporation State-changeable device
US10692577B2 (en) 2013-08-30 2020-06-23 International Business Machines Corporation State-changeable device
US10762962B2 (en) 2013-08-30 2020-09-01 International Business Machines Corporation State-changeable device

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