WO2021259963A1 - Immunization against wnt4 for treatment and prophylaxis of breast cancer - Google Patents

Abstract

The present invention relates to a method of preventing or treating breast cancer and/or metastases thereof in a female mammal. Specifically, the method comprises administering a pharmaceutical composition comprising an immunogen for inducing an immune response against a Wnt4 protein or an antibody or fragment thereof that specifically binds to the Wnt4 protein.

Description

Immunization against Wnt4 for treatment and prophylaxis of breast cancer

Field of the invention

The present invention relates to the fields of medicine, oncology, immunology, developmental biology and biopharmacy. More specifically, the invention relates to methods for treatment and prophylaxis of breast cancer, which method comprises the passive and/or active immunization against the Wnt 4 protein, using antigen sources providing at least an immunogenic portion of a Wnt 4 protein. Such antigen sources may be used in vaccines and pharmaceutical compositions for therapeutic and prophylactic treatment of primary and/or recurring breast cancer and metastases thereof.

Background of the invention

The lifetime absolute risk of women in the USA to develop breast cancer (BC) is 1 in 8 (12.4%) over an 80-year lifespan [1] For men, the lifetime risk of acquiring breast cancer is about 1 in 1 ,000 (0.1%) [2], which means that the risk in women is 125 times higher. Lifetime risk for women is similar in Europe, and even reported as 1 in 7 (14.3%) in the United Kingdom (UK) for women born after 1960, with a yearly incidence of new cases of about 480 per 100,000 for women, and about 10 per 100,000 for men [3] Geographical differences exist. In Japan, the lifetime risk of BC is lower with about 1 in 38 (2.6%) for women, but the huge difference with males remains with an incidence of about 1 in 2,000 (0.05%) in Japanese males [4]

Generally estrogens are considered to be responsible for causing BC. This perception is supported by the fact that estrogens are known to stimulate the growth of existing estrogen-receptor positive (ER+) BC indeed clinically as well as in preclinical in vitro and in vivo models [5] However, high dose estrogen (HDE) is also an effective treatment of BC more than 5 years after menopause or after the occurrence of resistance to endocrine anti-estrogen treatment [6].This contradictory knowledge is known as the “estrogen paradox” [5,7] Essential for HDE efficacy is an extended period of estrogen deprivation before the tumor is subjected to estrogen treatment (the “gap hypothesis”) [8,9] The stimulatory or inhibitory effect of estrogens on the growth of BC tumors must be distinguished from the question whether or not estrogens cause BC de novo [10] While estrogen metabolites are held responsible for causing BC, which may be true in in vitro and in vivo preclinical models [11], no evidence is available that women with BC have increased levels of such potentially carcinogenic metabolites in the general circulation or locally in the breast [12,13]

Many factors are known to be related to the risk of women to develop BC, including estrogen related risk factors such as breast density, estradiol (E2) levels, age at menopause, age at first life birth, age at menarche, body weight, obesity, menopausal hormone therapy and combined oral contraceptive use. Other non-estrogen related risk factors are a family history of BC and lifestyle related risks such as diet, alcohol use and exercise [14] Most factors are not relevant for males, but also for men, high E2 levels are related to a greater risk of developing breast cancer [15] Except for an early first pregnancy before the age of 20, which reduces the BC life time risk by about 50% in comparison to nulliparous women, all these factors affect the BC risk to a relatively small extent which is incomparable to the big gender difference. An exception is the presence of germline mutations, especially the BRCA1 or BRCA2 gene, which increases the BC risk with a factor of about 10 to a lifetime risk of 45-85% in women and 1 % in men [16-18]

So far, nobody has put the BC gender question in the perspective of some rare, but well defined human populations with major deviations of genetic, sexual and gender related structures and functions. We have searched in eight such populations for information on the occurrence and risk of BC in relation to gender, breast development, gonadal function and steroid hormone levels, especially estrogens, androgens and progesterone (P4).

The most surprising result of our analysis is that BC does not seem to occur in women with the Mayer-Rokitansky-Kiister-Hauser (MRKH) syndrome, who have normal female breasts and functioning ovaries with normal levels of female steroid hormones including progesterone (P4). On the basis of this finding, the cause of the MRKH syndrome, i.e. the absence of Wnt4 activity during embryonic development, is plausibly also responsible for the absence of BC later in life. Based on this finding, the present invention provides means and methods for immunization of females for the prophylaxis and therapy of BC in women.

Summary of the invention

The invention provides for a pharmaceutical composition comprising an immunogen for inducing an immune response against a Wnt4 protein or an antibody or fragment thereof that specifically binds to the Wnt4 protein for use in the treatment of breast cancer and/or metastases thereof. The treatment can be prophylactic i.e. before the onset of breast cancer or the treatment can be therapeutic to cure or delay existing breast cancer of metastases thereof.

Description of the invention Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

For purposes of the present invention, the following terms are defined below.

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

As used herein, the term "and/or" indicates that one or more ofthe stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... ,etc.

The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.

As used herein, "an effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a cancer varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

As used herein, the term "antibody" means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigen. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen. Antibodies can derive from multiple species. For example, antibodies include rodent (such as mouse and rat), rabbit, sheep, camel, and human antibodies. Antibodies can also include chimeric antibodies, which join variable regions from one species to constant regions from another species. Likewise, antibodies can be humanized, that is constructed by recombinant DNA technology to produce immunoglobulins which have human framework regions from one species combined with complementarity determining regions (CDRs) from a another species' immunoglobulin. The antibody can be monoclonal or polyclonal. Antibodies can be divided into isotypes (IgA, IgG, IgM, IgD, IgE, lgG1 , lgG2, lgG3, lgG4, lgA1, lgA2, lgM1 , lgM2). In another embodiment the term "antibody" refers to an intact antibody, or a fragment of an antibody that competes with the intact antibody for antigen binding. In certain embodiments, antibody fragments are produced by recombinant DNA techniques. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies. The term “antibody fragment” is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment of an anti-Wnt4 antibody is one that can bind to Wnt4. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and scFv. Exemplary antibody fragments also include, but are not limited to, domain antibodies, nanobodies, minibodies ((scFv-CH.sub.3).sub.2), maxibodies ((scFv-CH.sub.2--CH.sub.3).sub.2), diabodies (noncovalent dimer of scFv). The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.

Detailed description of the invention

The Mayer-Rokitansky-Kiister-Hauser (MRKH) syndrome or Miillerian agenesis is a congenital malformation characterized by failure of the Miillerian ducts to develop in an XX phenotype female, resulting in absence of the upper two-third of the vagina, the uterus, and the oviducts, all normally derived from the paramesonephric Miillerian ducts [19] Women with the MRKH syndrome have functioning ovaries, i.e. they ovulate and produce normal ovarian steroids, including progesterone (P4). Their major problem is infertility due to the absent uterus, but since they have oocytes pregnancy is possible by in-vitro fertilization (IVF) and the uterus of a surrogate mother and recently also by transplantation of a uterus.

The cause of MRKH is most likely the absence of activity of the growth factor Wnt4 during the critical period of formation of the internal genitalia 6-8 weeks after conception [20, 21] It has been demonstrated that the Miillerian duct fails to develop in the absence of Wnt4 [22] Wnt4 is a member of the Wnt family of secreted molecules that were originally identified as mammalian homologues of the Drosophila wingless gene. The Wnt4 gene on chromosome 1 and the encoded Wnt4 glycoprotein ligand (length 351 amino acids and mass 39.1 kDa) are signaling molecules shown to influence the sex-determination cascade known to antagonize the testis-determining factor and play a concerted role in both the control of female development and the prevention of testes formation. The Wnt4 gene, its nuclear receptor and the Wnt4 protein are associated with abnormal proliferation in breast tissue and have a special role in mammary development and in the origin and stimulation of breast cancer. Wnt4 is also required for normal antral follicle development and regulation of granulosa cell function including steroidogenesis [23] In addition, Wnt4 has an essential function in mammary gland development downstream of P4 signaling [24] In male embryo’s, Wnt4 activity is downregulated by the Sex-determining Region Y (SRY) gene on the Y- chromosome causing regression of the Miillerian ducts and development of the mesonephric Wolffian ducts, resulting in the formation of epididymis, vas deferens and seminal vesicles [25]

After thorough investigation, the inventors of present application surprisingly discovered that breast cancer (BC) does not occur in MRKH women. Furthermore, the inventors discovered that the low male BC risk can be explained by the absence of Wnt4 activity. These findings indicate that Wtn4 activity is an important cause of de novo BC in women and that downregulating the Wnt4 activity is therefore a relevant target for the (prophylactic) treatment of breast cancer and/or metastases thereof.

Accordingly, in one aspect, the invention provides for a pharmaceutical composition comprising an immunogen for inducing an immune response against a Wnt4 protein or an antibody or fragment thereof that specifically binds to the Wnt4 protein, for use in a method for therapeutic and/or prophylactic treatment of breast cancer and/or metastases thereof in a female mammal.

The term “breast cancer” as used herein refers to both primary and/or recurring breast cancers as well as metastases thereof that may have settled anywhere in the body. Preferably, the invention concerns prophylactic treatment of breast cancer i.e. preventing the occurrence of breast cancer and/or metastases thereof in a female mammal.

Breast cancers can be divided into at least five distinct molecular subtypes based on gene expression, cellular morphology, and response to treatment. Breast cancers can first be divided into two broad groups, estrogen-receptor (ER)-positive and ER-negative. These two groups can further be subdivided into additional distinct biologically and clinically significant subgroups. ER-positive tumors express estrogen-receptor, ER-responsive genes, and other proteins of luminal epithelial cells. Thus, ER-positive tumors are "luminal tumors," which can further be classified into luminal A and luminal B tumors, depending on the characteristic gene expression patterns.

ER-negative tumors can be further classified into three groups: HER-2 positive, basal-like tumors, and normal breast-like tumors. HER-2 positive tumors express high levels of genes located in the HER2 amplicon on chromosome 17 at location 17q21 , including HER-2 and growth factor receptor-bound protein 7 (GRB7). They also have a high level of nuclear factor (NF)-kappaB activation and express a high level of the transcription factor GATA4 but lack expression of ER and GATA3. The normal breast-like tumors resemble normal breast tissue samples with relatively high expression of many genes characteristic of adipose cells and other nonepithelial cell types, and low levels of expression of luminal epithelial cell genes.

Basal-like breast cancers (BLBCs) are defined by the lack of expression of the hormone receptors estrogen (ER) and progesterone (PR), and the human epidermal growth factor receptor- 2 (HER2).

By "triple negative breast cancer" is meant estrogen-receptor (ER) negative, progesterone- receptor (PR) negative, and HER2 negative breast cancer. Triple negative breast cancers do not express ER, PR, or HER2.

The highest Wnt activity is found in basal-like BC, triple-negative BC, and more specific in subsets of BC cancer stem cells.

As used herein the terms "immunogen" or "immunogenic agent" or "antigen" are used interchangeably to describe a molecule capable of inducing an immunological response against itself on administration to a recipient, either alone, in conjunction with an adjuvant, or presented on a display vehicle.

As used herein the phrase "immune response" or its equivalent "immunological response" refers to the development of a humoral (antibody mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against a protein in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody, antibody containing material, or primed T-cells. Accordingly, in a preferred embodiment the invention relates to a method for therapeutic and/or prophylactic treatment of breast cancer and/or metastases thereof in a female mammal by active immunization. The method of active immunization preferably comprises administering a source of an immunogenic polypeptide capable of eliciting a cellular immune response against Wnt4 and/or a humoral immune response against Wnt4.

In a preferred embodiment the immunogenic polypeptide comprises a contiguous amino acid sequence selected from the amino acid sequence of an Wnt4 protein, which contiguous amino acid sequence preferably comprises at least one of a B cell epitope class I MHC- and a class II MHC- restricted T cell epitope.

Preferably, the immunogenic (poly)peptide comprises a B-cell epitope. The B-cell epitope or epitopes may be of any chemical nature, including without limitation peptides, carbohydrates, lipids, glycopeptides and glycolipids. In particular embodiments, the epitopes are peptides derived from one or more of the antigens described herein or known in the art. The epitope may be identical to a naturally occurring epitope, or may be a modified form of a naturally occurring epitope. B-cell epitopes are recognized by B-cells and by antibodies. B-cell epitopes are typically at least five amino acids, more often at least six amino acids, still more often at least seven or eight amino acids in length, and may be continuous ("linear") or discontinuous ("conformational"); the latter being formed, for example, by the folding of a protein to bring non-contiguous parts of the primary amino acid sequence into physical proximity. Conformational epitopes may be recognizable as linear epitopes, such as when they are synthetically prepared as such. Typically, linear B-cell epitopes vary from 5-20 amino acids in length. B-cell epitopes may also be carbohydrate epitopes.

Experimental methods to identify B-cell epitopes are known in the art. Generally, there are two important aspects to consider when identifying B-cell epitopes. First is the primary amino acid sequence of the protein. Second is the conformational structure of the protein.

The antigen of the compositions described herein may either consist of or comprise a B-cell epitope capable of inducing a humoral immune response.

In some embodiments, the immunogen of the compositions described herein may consist of or comprise one or more B-cell epitopes associated with an infectious disease. For example, the immunogen may consist of or comprise a B-cell epitope derived from a virus, such as for example influenza virus, Zika virus or respiratory syncytial virus. In an embodiment, the B-cell epitope may be an epitope derived from the hemagglutinin glycoprotein of the H5N1 influenza virus. In another embodiment, the B-cell epitope may be an epitope derived from the ectodomain of the small hydrophobic protein (SHe) of a respiratory syncytial virus.

In another embodiment, the antigen of the compositions described herein may consist of or comprise a B-cell epitope derived from a bacterium, such as for example Bordetella pertussis or Bacillus anthracis. In an embodiment, the B-cell epitope may be an epitope of the pertussis toxoid protein produced by Bordetella pertussis. In another embodiment, the B-cell epitope may be an epitope of the anthrax recombinant protective antigen (rPA) or the anthrax mutant recombinant protective antigen (mrPA). ln another embodiment, the antigen of the compositions described herein may consist of or comprise a B-cell epitope derived from aprotozoan, such as from the genus Plasmodium.

In a further embodiment, the composition may comprise a mixture of B-cell epitopes as antigens for inducing a humoral immune response. The B-cell epitopes may be linked to form a single molecule (e.g. one polypeptide) or be presented as separate molecules (e.g. separate polypeptides).

More preferably, the immunogenic polypeptide comprises a contiguous amino acid sequence selected from the amino acid sequence of the Wnt4 protein (i.e. SEQ ID NO:1 or the amino acid sequence of GenBank accession number AL031281 .6) which contiguous amino acid sequence preferably comprises at least one of B-cell epitope, a class I MHC- and a class II MHC-restricted T cell epitope. More preferably, the contiguous amino acid sequence comprises at least one of a class I MHC- and a class II MHC-restricted T cell epitope with a low percentile rank (see Moutaftsi et al., Nat Biotechnol. 2006 Jul;24(7):817-9; and Kotturi et al., J Virol. 2007 May;81 (10):4928-40). A class I MHC-restricted T cell epitope with a low percentile rank preferably is an epitope with a percentile rank that is not higher than 1.00, 0.80, 0.40, 0.30, 0.20, 0.15, 0.10 or 0.05 (see e.g. Tables 2 and 5). A class II MHC-restricted T cell epitope with a low percentile rank preferably is an epitope with a percentile rank that is not higher than 2.50, 2.40, 2.05, 2.00, 1 .80, 1 .60, 1 .40, 1 .20, 1.10, 1 .00, 0.90, 0.70, 0.60, 0.50, 0.40, 0.20. 0.15, 0.10, 0.05 or 0.02.

The contiguous amino acid sequence from an Wnt4 protein as comprised within the immunogenic polypeptide, preferably comprises an immunologically active (sequence) fragment of the Wnt4 protein. The term “immunologically active fragments thereof will generally be understood in the art to refer to a fragment of a Wnt4 protein antigen comprising at least an epitope, which means that the immunogenic polypeptide at least comprises 4, 5, 6, 7 or 8 contiguous amino acids from the sequence of the Wnt4 protein antigen. According to the present invention the fragment comprises at least an MHC class I or MHC class II binding peptide presented by such MHC molecule to the immune system. An ‘immunologically active fragment’ according to this invention comprises at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or 17 contiguous amino acids from the sequence of the Wnt4 protein antigen or homologue or analogue thereof. While the majority of the MHC binding peptides are of a length of 9 amino acids, longer peptides can be accommodated by the bulging of their central portion (Guo et al., 1992, Nature; 360(6402):364-366; Speir et al., 2001 , Immunity; 14(1):81— 92), resulting in binding peptides of length 8 to 15 (Schumacher et al., 1991 , Nature; 350(6320):703-706). Peptides binding to class II proteins are not constrained in size (Nelson et al., 1999, Rev Immunogenet; 1 (1 ):47— 59; Yassai et al., 2002, J Immunol; 168(3):1281 — 1285) and can vary from 11 to 30 amino acids long (Rammensee 1995, Immunogenetics; 41 (4): 178-228) possibly even whole proteins. The binding motif however is about 9 amino acids long. MHC class II can accommodate much longer peptides than MHC class I because the ends of the MHC II binding groove are open, hence an epitope (binding into the groove) may be flanked by additional stretches of amino acids on either end. Still more preferably the fragment comprises both a Cytotoxic T Lymphocyte (CTL) and a Helper T Lymphocyte (HTL) epitope. Most preferably however, the fragment is a peptide that requires processing by an antigen presenting cell (APC), i.e. the fragment has a length of at least about 18 amino acids, which 18 amino acids are not necessarily a contiguous sequence from the Wnt4 protein antigen.

The length of a contiguous amino acid sequence from Wnt4 protein as comprised within the immunogenic polypeptide or the length of the immunogenic polypeptide itself, therefore preferably is at least 18, 19, 20, 21 , 22, 25, 27, 30, 33 or 35 amino acids and preferably no more than 100, 80, 60, 50, 45, 40, 35, 33 or 30 amino acids. Preferably the length of a contiguous amino acid sequence from an Wnt4 protein as comprised within the immunogenic polypeptide, or the length of the immunogenic polypeptide itself, is 19 - 50 or 19 - 45, more preferably 25 - 40 amino acids, even more preferably 25 - 35 and most preferably 25 - 30 amino acids. From the view point of manufacturability an immunogenic polypeptide with a length of around 25 amino acids is optimal, while still long enough to contain multiple epitopes and force presentation via Antigen Presenting Cells.

Most preferably, the immunogenic polypeptide comprises a peptide comprising a contiguous amino acid sequence of 5 - 25 amino acids that comprises a B cell epitope. Preferably, this B-cell epitope is specific for Wnt4, i.e. selected from a region that does not comprise conserved amino acid sequences of the other Wnt proteins.

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

A homologue herein is understood to comprise an immunogenic polypeptide having at least 70 %, preferably at least 80 %, more preferably at least 90 %, still more preferably at least 95 %, still more preferably at least 98 % and most preferably at least 99% amino acid sequence identity with the naturally occurring Wnt4 polypeptide mentioned above and is still capable of eliciting at least the immune response obtainable thereby. A homologue or analogue may herein comprise substitutions, insertions, deletions, additional N- or C- terminal amino acids, and/or additional chemical moieties, such as carbohydrates, to increase stability, solubility and immunogenicity.

Expressly included in the invention is the use of an immunogenic polypeptide comprising an amino acid sequence that is a homologue of a wild-type Wnt4 protein, but differs therefrom as a result of tumor-specific mutations (which can be patient-specific or shared) that result in altered amino acid sequences, i.e. so-called neoantigens.

The term "epitope" as used herein refers to a portion of an antigen, typically defined by a short peptide, which is capable of eliciting a cellular or humoral immune response when presented in a physiologically relevant context in vivo. A "T cell epitope" refers to a short peptide or portion thereof that binds to an MHC molecule and is recognized by certain T cells when presented in certain MHC molecules. A T cell epitope is capable of inducing a cell mediated immune response via direct or indirect presentation in heterodimeric membrane MHC molecules. For the sake of clarity, the peptide of the invention preferably comprises at least one MHC class I presented epitope and preferably also at least one MHC class II presented epitope. Each of these epitopes are presentable and will bind to the corresponding specific MHC molecule present on the cells after having been processed as described herein. Each MHC restricted epitope may therefore also be named an MHC binding and/or presentable epitope. Preferably, a specific proteasomal cleavage site generating the C-terminus of such epitope is present exactly after the epitope’s amino acid sequence in order to be liberated from the immunogenic polypeptide and presented on the MHC class I molecule. Length requirements are much less strict for MHC class II presented epitopes, therefore a need for precise enzymatic generation of the class II binding peptide is less absolute. Briefly, MHC molecules preferentially bind particular amino acid residues known as "anchor" residues (K. Falk et al., Nature 351 :290-96 (1991)). This characterization permits class I and II MHC binding motifs to be recognized within any known peptide sequence (see e.g. Tables 2, 5 and 3).

In the present context, the term "MHC restricted epitope" is synonymous with T cell epitope. The term "class I MHC restricted epitope", as used herein, refers to peptide sequences recognized by cytotoxic T lymphocytes (also called CD8+ cells, TCD8 or CTLs) in association with class I MHC. The term "class II MHC restricted epitope", as used herein, refers to a peptide recognized by helper T cells (also called CD4+ cells, TCD4 or HTLs) in association with class II MHC. A “B cell epitope" is the portion of an antigen that is capable of binding to an antigen binding site of an immunoglobulin and therefore capable of stimulating a humoral response without presentation by an MHC molecule. As explained herein before the polypeptide useful in the present invention, or the nucleic acid encoding said polypeptide, comprises at least one T cell epitope. The use of polypeptides that also comprise a B cell epitope is however not excluded from the present invention. The present immunogenic polypeptides may also include multiple T cell epitopes and, optionally a B cell epitope. When multiple epitopes are present in a peptide, the epitopes may be oriented in tandem or in a nested or overlapping configuration wherein at least one amino acid residue may be shared by two or more epitopes. The immunogenic polypeptide of the invention preferably includes one or more MHC class I restricted epitopes. As is generally known by the skilled person, an antigen comprising a single MHC restricted epitope will be useful only for treating a (small) subset of patients who express the MHC allele product that is capable of binding that specific peptide. It has been calculated that, in humans, vaccines containing CTL epitopes restricted by HLA-A1 , -A2, -A3, -A24 and -B7 would offer coverage to approximately 80 % of individuals of most ethnic backgrounds. Therefore, if the present method is used to treat a human, it is particularly preferred that the method comprises the administration of a composition comprising one or more different polypeptides comprising one, more preferably two, most preferably three MHC class I binding native Wnt4 epitopes selected from HLA-A1 , HLA-A2, HLA-A3, HLA-A24 and HLA-B7 restricted epitopes; or homologues thereof.

According to another embodiment the immunogenic polypeptide of the invention preferably includes one or more MHC class II restricted epitopes. The most frequently found MHC class II allele products in humans include HLA-DR1 , -DR3, -DR4 and -DR7. Accordingly, it is preferred that the method comprises the administration of a composition comprising one or more different polypeptides, said one or more different polypeptides comprising one, more preferably two and most preferably three MHC class II binding native Wnt4, epitopes selected from HLA-DR1 , HLA- DR3, HLA-DR4 and HLA-DR7 restricted epitopes; or homologues thereof.

In still another embodiment, the method of the invention comprises the administration of a composition comprising one or more polypeptides, said one or more polypeptides comprising one or more MHC class I restricted epitopes and one or more MCH class II restricted epitopes or homologues thereof. Even, more preferably said composition comprises an effective amount of one or more different polypeptides that together include essentially all of the MHC class I and MHC class II binding epitopes comprised in Wnt4, glycoproteins; or homologues of said one or more polypeptides. In one embodiment, the present method comprises the administration of a composition comprising one or more different immunogenic polypeptides. Preferably, said one or more different polypeptides together comprise at least 50 %, more preferably at least 70 %, still more preferably at least 80 %, still more preferably at least 90 % and most preferably at least 95 % of the MHC class I and MHC class II restricted epitopes comprised in Wnt4 or homologues of said one or more polypeptides.

In a preferred embodiment, the pharmaceutical composition of the invention comprises a T cell comprising a T cell receptor that binds an MHC-peptide complex, wherein the peptide is a peptide from the amino acid sequence of the Wnt4 protein. Such a T cell can e.g. be obtained in a method comprising contacting a T-cell with an antigen presenting cell expressing a polynucleotide encoding an immunogenic polypeptide of the invention and/or contacting a T-cell with an antigen presenting cell loaded with an immunogenic polypeptide of the invention; and, optionally, culturing said T-cell. The antigen presenting cell (APC), preferably is a dendritic cell (DC). The T-cell is preferably a CD8+ cytotoxic T-cell or a CD4+ T-helper cell. Introducing a polynucleotide encoding the immunogenic polypeptide into the APC or DC may be performed using any method known to the person skilled in the art, preferably a polynucleotide according to the invention is introduced into the APC or DC using transfection. Preferably the polynucleotide encoding the immunogenic polypeptide is provided with proper control sequences, or be comprised in a proper expression vector. Contacting a T-cell with an immunogenic polypeptide of the invention can be performed by any method known to the person skilled in the art. Preferably, the immunogenic polypeptide or an epitope comprised in the immunogenic polypeptide is presented to the CD8+ cytotoxic T-cell or CD4+ T-helper cell by an MHC class I or an MHC class II molecule on the surface of an APC, preferably a DC. The person skilled in the art knows how to load an APC or DC with a peptide. Culturing said T-cell may be performed using any method known by the person skilled in the art. Maintaining a T-cell under conditions to keep the cell alive is herein also to be construed to be culturing. Preferably, the T-cell according to this aspect of the invention is contacted with an immunogenic polypeptide according to the invention as defined in the first aspect of the invention. Ex vivo methods for obtaining and activating tumor antigen-specific T-cells are described in more detail e.g. in WO2017/173321 . In this aspect the invention also relates to a composition comprising an (activated) T-cell according to the invention, as well as to methods of the inventions for therapeutic and/or prophylactic treatment of breast cancer and/or metastases, comprising administering to the subject a therapeutically effective amount of an (activated) tumor specific T- cell described herein. In silico prediction software, such as NETMHCPAN 3.0 can be used to predict potential HLA-A2 restricted T-cell epitopes from hWnt4. In embodiments, the administering comprises administering from about 106 to 1012, from about 108 to 1011 or from about 109 to 1010 of the (activated) tumor specific T-cells. The T-cell or composition therewith is preferably administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration. In another embodiment, the T-cell or composition therewith is administered into an anatomic site that drains into a lymph node basin. In another embodiment, the administration is into multiple lymph node basins.

In one embodiment, the pharmaceutical composition for a use according to the invention comprises a source of the immunogenic polypeptide. The source of the immunogenic polypeptide can be a proteinaceous source, a nucleic acid, an antigen presenting cell or a combination thereof. The proteinaceous source can e.g. be a composition comprising one or more peptides, polypeptides or proteins that act as immunogen.

Preferably, the source of the immunogenic polypeptide of the invention to be administered comprises a nucleic acid molecule encoding the immunogenic polypeptide. The source or composition comprising the nucleic acid molecule encoding the immunogenic polypeptide can comprise one or more different nucleic acid molecule encoding any one of the immunogenic polypeptides, polypeptide fragments, and/or peptides as herein defined above. In addition, the nucleic acid molecule can encode a larger part of a native Wnt4. The nucleic acid molecule can e.g. encode a polypeptide comprises at least 50, 70, 80, 90, 95 or 100% of the complete amino acid backbone of a Wnt4, preferably a human Wnt4, more preferably of hWnt4(23-351) or a homologue of said polypeptide. Preferably the nucleic acid molecule encodes a contiguous stretch of at least 50, 70, 80, 90, 95 99% or 100%of the complete amino acid backbone. It is also possible that a nucleic acid molecule encodes more than one (T-cell epitope containing) immunologically active fragments of contiguous amino sequences from an Wnt4 as defined hereinabove, whereby in the encoded amino acids sequences, the different immunologically active fragments can be separated by spacer or linker sequences as beads on a string.

The nucleic acid molecule encoding the immunogenic polypeptide of the invention can be a DNA molecule, preferably a genetic construct wherein the nucleotide sequence coding for the immunogenic polypeptide (cDNA) is operably linked to appropriate expression regulatory sequence that ensure functional expression of the immunogenic polypeptide in the target cells in the human subject, e.g. including at least a strong (e.g. viral) promoter. Genetic constructs for use as DNA vaccines, including e.g. plasmids or viral vectors, are inter alia described in WO2014/165291 , WO2016/123285 and WO2017/136758. Alternatively, the nucleic acid molecule encoding the immunogenic polypeptide of the invention can be an RNA molecule, e.g. an mRNA, ssRNA, dsRNA or combinations thereof. The RNA molecule can e.g. be formulated in a particle comprising the molecule. Suitable embodiments for RNA-based vaccines are e.g. described in WO2013/087083.

In one embodiment, the source of the immunogenic polypeptide of the invention can further be a live cell that expresses and/or presents the immunogenic polypeptide. The cell can be an autologous or allogeneic immune cell, e.g. a dendritic cell derived from the subject to treated, or the cell can be a microbial cell, more preferably a bacterium such as e.g. a live-attenuated Listeria monocytogenes. The expressed immunogenic polypeptide preferably is an immunogenic polypeptide as defined hereinabove. The immunogenic polypeptide can be expressed as part of a fusion protein, wherein preferably the immunogenic polypeptide is fused to a protein that is endogenous to the organism, e.g. an N-terminal fragment of an L. monocytogenes LLO or ActA protein. Suitable embodiments for Listeria-based vaccines are e.g. described in WO2015/164121. A bacterium expressing the immunogenic polypeptide of the invention can be administered orally or parenterally, preferably intravenously. In another embodiment of the invention, the source of the immunogenic polypeptide of the invention is an autologous or allogeneic dendritic cell (DC) that presents at least one MHC restricted epitope of the immunogenic polypeptide in an HLA molecule on its surfaces. Such dendritic cells can e.g. be prepared ex vivo by contacting and/or loading DCs from the patient’s blood, e.g. DCs isolated from mononuclear cells from the patient/subject, with a composition comprising the immunogenic polypeptide of the inventions. The immunogenic polypeptide contacted with mononuclear cells or DCs preferably is an immunogenic polypeptide as defined hereinabove. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DCs with peptides and washing to remove unbound peptides, the DCs are reinfused into the patient. In this embodiment, a composition is provided comprising peptide-pulsed DC which present the pulsed peptide epitopes in HLA molecules on their surfaces. Alternatively, instead of using autologous cells derived from the subject, allogenic DCs can be used that are derived from a precursor human dendritic cell line and designed to deliver tumor associated antigens, such as e.g. described in W02009/019320, WO2014/090795 and WO2014/006058. Methods of inducing an immune response employing ex vivo peptide-pulsed DC are well known to the skilled person. For immunization purposes the aforementioned immunogenic polypeptides of the invention may also be fused with a carrier molecule. Preferably the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae. In one embodiment the pharmaceutical composition for a use according the invention, is for the treatment and/or prophylactic treatment of a human female. Preferably, the female mammal is a human female and the Wnt4 protein is a human Wnt4 protein.

In one embodiment of the invention, the human Wnt4 protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of an allelic variant thereof having at least 95 % sequence identity with SEQ ID NO: 1. Preferably the human Wnt4 protein comprises an amino acid sequence with at least 95 % sequence identity with the amino acid sequence of the mature Wnt4 protein, comprises positions 23-351 of SEQ ID NO: 1.

A further aspect of the invention relates to a method for therapeutic and/or prophylactic treatment of breast cancer and metastases thereof in a subject by administering an antibody or fragment thereof that specifically binds to an epitope of human Wnt4 protein. Preferably, the antibody is a humanized or human monoclonal antibody. More preferably, the antibody or fragment thereof specifically binds to an epitope of human Wnt4 or human Wnt4(23-351).

An antibody "which binds" an antigen of interest, e.g. a tumor-associated hWnt4 protein antigen or epitope thereof, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoassay (RIA). With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labelled target. In this case, specific binding is indicated if the binding of the labelled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target (which may be determined as described below) of at least about 10-4 M, alternatively at least about 10-5 M, alternatively at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

A "Kd" or "Kd value" can be measured by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™- 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~10 - 50 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10mM sodium acetate, pH 4.8, into 5 pg/ml (-0.2 pM) before injection at a flow rate of 5pl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of the antibody or Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25pl/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensorgram. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds 106 M-1 S-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette. An "on-rate" or "rate of association" or "association rate" or "kon" according to this invention can also be determined with the same surface plasmon resonance technique described above using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) as described above. One embodiment of the invention concerns a method of treating method for therapeutic and/or prophylactic treatment of breast cancer and/or metastases thereof, the method comprising administering to said subject a composition comprising an anti-Wnt4 antibody, preferably an anti- hWnt4 or anti-hWnt4(23-351) antibody, wherein the antibody induces killing of breast cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC) or apoptosis. As is generally understood by those of average skill in the art these antibody effector functions may be mediated by the Fc portion of the antibody, e.g. by binding of an Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity or by binding of an Fc effector domain(s) to components of the complement system. Typically, the effect(s) mediated by the Fc-binding cells or complement components eventually result in inhibition and/or depletion of target cells, i.e.Wnt4- expressing cells. Human IgG isotypes lgG1 , lgG2, lgG3 and lgG4 exhibit differential capacity for effector functions. ADCC may be mediated by lgG1 and lgG3, ADCP may be mediated by lgG1 , lgG2, lgG3 and lgG4, and CDC may be mediated by lgG1 and lgG3. In the methods described herein the anti-hWnt4 or anti-hWnt4(23-351) antibody preferably is an lgG1 , lgG2, lgG3 or lgG4 antibody.

For example, the antibody for a use according to the invention is a humanized version of the anti-Wnt4 mouse monoclonal antibody ab169592 (Abeam) or B- 6 (sc376279; Santa Cruz Biotechnology). Humanization of non-human (e.g., rodent) antibodies is described in Jones et al. , Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1 :105-115 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994). Commercial humanization of non-human antibodies is offered by companies such as www.promab.com, www.genscript.com and www.oakbiosciences.com. Alternatively, anti-Wnt4 monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), e.g. using full length human Wnt4 protein as immunogen, as described by www.abcam.com (see product datasheet Anti-Wnt4 antibody ab169592). Alternatively, anti-Wnt4 monoclonal antibodies may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). .

In the methods of the invention described herein the anti-Wnt4 antibody may be provided in suitable pharmaceutical compositions comprising the anti-Wnt4 antibody and a pharmaceutically acceptable carrier. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

In certain embodiments, the pharmaceutical composition of the invention is administered to a human female prior to menarche. In certain embodiments the pharmaceutical composition of the invention is administered post-menarche. In certain embodiments the pharmaceutical composition of the invention is administered to a human female before menopause. In certain embodiments, the pharmaceutical composition of the invention is administered to a human female between the ages of 11-55. In certain embodiments, the pharmaceutical composition of the invention is for the prophylactic treatment of breast cancer. The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably. In one embodiment, the pharmaceutical composition of the invention is for the prophylactic treatment of breast cancer of a human female prior to menarche.

In one embodiment, the pharmaceutical composition of the invention is administered concurrently with HPV vaccination. In one embodiment, the pharmaceutical composition of the invention is for the prophylactic treatment of breast cancer in a human female at risk to develop breast cancer. By "being at risk" is meant a subject that is identified as having a higher than normal chance of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had, or who currently has, a disease, in particular cancer, is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease. Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases. In one embodiment, the human female is at risk to develop breast cancer due to a BRCA1 or BRCA2 mutation.

In one embodiment, the pharmaceutical composition for a use according to the invention is for use in the therapeutic treatment of breast cancer. “Therapeutic treatment” is herein defined as curing, alleviating or delaying the progression of breast cancer and or metastases thereof.

The present method preferably comprises administration of the present immunogenic polypeptides and compositions comprising them via the parenteral or oral route, preferably the parenteral route. Preferred routes of administration include, but are not limited to, intratumoral, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intra-arterial, intraocular and oral as well as topically, transdermal, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. In another embodiment, administration is into an anatomic site that drains into a lymph node basin. In another embodiment, the administration is into multiple lymph node basins.

Examples

Example 1 : WNT-4 expression in MRKH patients.

Biopsies from breast tissue are taken from four women diagnosed with MRKH and from control women during the mid-luteal phase of the menstrual cycle to ensure that progesterone levels are at their peak. In parallel to the biopsy, blood samples from these women are taken for analysis of the hormonal levels at the time of the biopsies. The genomic and transcriptomic material is isolated from the breast biopsies using DNA and RNA isolation and purification techniques that are known in the art. The isolated genomic and transcriptomic material is subjected to next generation sequencing (NGS) using a library of oligonucleotide probes to enable a genome wide DNA mutation analysis and RNA sequencing of the sample. Wnt-4 expression is observed to be reduced or absent in the four MRKH patients used in this study. References

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Claims

Claims

1 . A pharmaceutical composition comprising an immunogen for inducing an immune response against a Wnt4 protein or an antibody or fragment thereof that specifically binds to the Wnt4 protein, for use in a method for therapeutic and/or prophylactic treatment of breast cancer and/or metastases thereof in a female mammal.

2. Pharmaceutical composition for use according to claim 1 , wherein the composition comprises: a) a source of an immunogenic polypeptide comprising at least one of a B cell epitope, a class I MHC- and a class II MHC-restricted epitope from the amino acid sequence of the Wnt4 protein; or, b) a T cell comprising a T cell receptor that binds an MHC-peptide complex, wherein the peptide is a peptide from the amino acid sequence of the Wnt4 protein.

3. Pharmaceutical composition for a use according to claim 2, wherein the source of the immunogenic polypeptide comprises at least one of: a) a proteinaceous composition comprising at least one immunogenic polypeptide as defined in claim 2; b) a nucleic acid molecule encoding an amino acid sequence of said immunogenic polypeptide; c) a cell expressing said immunogenic polypeptide; and, d) an antigen presenting cell presenting an MHC-peptide complex wherein the peptide is a peptide from the amino acid sequence of an Wnt4 protein.

4. Pharmaceutical composition for a use according to claim 3, wherein: a) the immunogenic polypeptide comprises a contiguous amino acid sequence of at least 5 amino acids selected from the amino acid sequence of human Wnt4 protein and wherein the contiguous amino acid sequence comprises at least one a B cell epitope, a class I MHC- and a class II MHC-restricted epitope; b) the nucleic acid molecule is: i) a DNA molecule that is an expression construct for expression of the immunogenic polypeptide in a human cell; or, ii) an RNA molecule that is capable of being translated into the immunogenic polypeptide in a human cell; c) the cell is a microbial cell, preferably a Listeria cell; and, d) the antigen presenting cell is an autologous or allogeneic dendritic cell that is loaded ex vivo with an immunogenic polypeptide as defined in claim 2.

5. Pharmaceutical composition for a use according to any one of claims 2 - 4, wherein the immunogenic polypeptide comprises a peptide comprising a contiguous amino acid sequence of 5 - 25 amino acids selected from the amino acid sequence of a WNT4 protein, wherein preferably the peptide is couple to a carrier protein, wherein preferably the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA),

E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

6. Pharmaceutical composition for a use according to any one of the preceding claims, wherein the female mammal is human and the Wnt4 protein is a human Wnt4 protein.

7. Pharmaceutical composition for a use according to claim 6, wherein the human Wnt4 protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of an allelic variant thereof having at least 95 % sequence identity with SEQ ID NO: 1 , preferably the human Wnt4 protein comprises an amino acid sequence with at least 95 % sequence identity with the amino acid sequence of the mature Wnt4 protein, comprises positions 23-351 of SEQ ID NO: 1.

8. Pharmaceutical composition for a use according to claim 1 , wherein the pharmaceutical composition comprises a humanized or human monoclonal antibody or fragment thereof that specifically binds to human Wnt4 protein.

9. Pharmaceutical composition for use according to any one of the preceding claims, wherein the pharmaceutical composition is used in the prophylactic treatment of breast cancer.

10. Pharmaceutical composition for use according to claim 9, wherein the pharmaceutical composition is used in the prophylactic treatment of breast cancer prior to menarche.

11. Pharmaceutical composition for use according to claim 10, wherein the pharmaceutical composition is administered concurrently with HPV vaccination

12. Pharmaceutical composition for use according to claim 11 , wherein the pharmaceutical composition is used in the prophylactic treatment of breast cancer in human females with an increased risk of developing breast cancer due to a BRCA1 or BRCA2 mutation.

13. Pharmaceutical composition for use according to any one of claims 1-8, wherein the pharmaceutical composition is used in the therapeutic treatment of breast cancer.

14. Pharmaceutical composition for a use according to anyone of the preceding claims, wherein the method of treatment comprises administration of the pharmaceutical composition by at least one of intratumoral, intramuscular, intraperitoneal, intradermal, subcutaneous and transdermal routes and/or into an anatomic site that drains into at least one lymph node basin.