WO2025114381A1

WO2025114381A1 - Muc1 antibodies and uses thereof

Info

Publication number: WO2025114381A1
Application number: PCT/EP2024/083802
Authority: WO
Inventors: Joseph Verdi; Fotini Papavasiliou-Stebbins; Monica Chandra; Charles Erec Stebbins; Sebastian Kruse; Katharina URBAN; Anastasia GKEKA; Paraskevi Maria VLACHOU-EFSTATHIOU; Eirini SIDIROPOULOU
Original assignee: Panosome GmbH; Deutsches Krebsforschungszentrum DKFZ
Current assignee: Panosome GmbH; Deutsches Krebsforschungszentrum DKFZ
Priority date: 2023-11-27
Filing date: 2024-11-27
Publication date: 2025-06-05
Anticipated expiration: 2026-05-27

Abstract

The present invention concerns the field of antibodies. More specifically, it relates to an antibody that specifically binds to hypoglycosylated MUC1, wherein said antibody binds to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein. The present invention also relates to said antibody for use in in treating and/or preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma. Moreover, the present invention refers to VAST bound immunogen for use as a vaccine, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP) wherein sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond. The invention also relates to method for manufacturing an antibody that specifically binds to hypoglycosylated MUC1, said method comprising the step of obtaining the antibody from a sample of an animal which has been immunized with a VAST-bound immunogen, wherein said immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Description

MUC1 antibodies and uses thereof

The present invention concerns the field of antibodies. More specifically, it relates to an antibody that specifically binds to hypoglycosylated MUC1, wherein said antibody binds to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein. The present invention also relates to said antibody for use in in treating and/or preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma. Moreover, the present invention refers to a VAST bound immunogen for use as a vaccine, wherein said immunogen is a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP) wherein sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond. The invention also relates to method for manufacturing an antibody that specifically binds to hypoglycosylated MUC1, said method comprising the step of obtaining the antibody from a sample of an animal which has been immunized with a VAST-bound immunogen, wherein said immunogen is a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Mucins are high molecular weight glycoproteins that are produced by epithelial tissues in most animals and their main function is to lubricate epithelial cell surfaces as well as protect them against invading pathogens. Mucins can be divided into three distinct subfamilies: (i) secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, MUC6, and MUC19, as protective barriers for underlying mucosal cells), (ii) cell-surface mucins (MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, and MUC20), and (iii) secreted non-gel-forming mucins (MUC7) (S.K. Linden et cd.. Mucosal Immunol. 208).

Mucin 1 (MUC1) is the most extensively studied among the mucins as it is the most ubiquitously expressed across all mucosal tissues. It is a single pass type I transmembrane heterodimer, comprised of two subunits: an a-subunit consisting of an extracellular N-terminal domain and a P-subunit composed of a transmembrane (TM) helix and cytoplasmic tail (CT). The extracellular domain of MUC1 contains a variable number of tandem repeats (VNTR) consisting of 20 to 120 repeats of a 20-amino acid sequence (HGV7iS'APD7RPAPGA7APPA) (SEQ ID NO: 93). The hyperglycosylated extracellular N-terminal domain extends up to 200- 500 nm from the cell surface. MUC1 is mainly expressed on the surface of glandular or epithelial cells of almost all tissues (M. Bose and P Mukherjee, Vaccines 2020). During translation, MUC1 is cleaved, and the extracellular domain including 20 to 120 variable number tandem repeats is bound to the membrane by noncovalent interaction with the C-terminal domain of MUC1 that consists of a short extracellular domain, the transmembrane domain and the cytoplasmic domain (T. Stasyk et al., J. Cell. Biochem. 2016). These repeats are rich in serine, threonine and proline residues which permits heavy O- and N-glycosylation.

Due to its overexpression and aberrant glycosylation with specific truncated carbohydrates in many epithelial cancers, MUC1 is an attractive target for immunotherapy (S.K. Lau etal., Am. J. Clin. Pathol. 2004). In particular, antibody-based immunotherapy has been used for cancer treatment for the past two decades and is one of the most effective ways to treat hematological malignancies and solid tumors (L.M. Weiner et al., Cell 2012). In recent years, various efforts have been made to generate effective anti-MUCl antibodies using the full-length MUCl/transmembrane domain (TM) molecule as immunogen (D.B. Rubinstein et al., Int J Cancer 2009) and numerous monoclonal anti-MUCl antibodies are in clinical trials or under pre-clinical or experimental studies. However, inadequate cancer cell selectivity as well as poor affinity and specificity has been reported for many of these anti-MUCl antibodies resulting in serious adverse effects due to their off-targeting of healthy cells. A further major drawback is that immunization with whole MUC1/TM molecule invariably resulted in an antibody response composed almost in its entirety of antibodies recognizing epitopes on the highly immunogenic tandem repeat array.

Hence, there is a need of more anti-MUCl antibodies with high specificity and reduced off- target effects, as well as methods of manufacturing such antibodies.

The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

Thus, the present invention relates to an antibody that specifically binds to hypoglycosylated MUC1, wherein said antibody binds to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein.

It is to be understood that in the specification and in the claims, “a” or “an” can mean one or more of the items referred to in the following depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that at least one item can be utilized. As used in the following, the terms “have”, “comprise” or “include” are meant to have a nonlimiting meaning or a limiting meaning. Thus, having a limiting meaning these terms may refer to a situation in which, besides the feature introduced by these terms, no other features are present in an embodiment described, i.e. the terms have a limiting meaning in the sense of “consisting of’ or “essentially consisting of’. Having a non-limiting meaning, the terms refer to a situation where besides the feature introduced by these terms, one or more other features are present in an embodiment described.

Further, as used in the following, the terms “preferably”, “more preferably”, “most preferably”, "particularly", "more particularly", “typically”, and “more typically” are used in conjunction with features in order to indicate that these features are preferred features, i.e. the terms shall indicate that alternative features may also be envisaged in accordance with the invention.

Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one item shall be used this may be understood as one item or more than one item, i.e. two, three, four, five or any other number. Depending on the item the term refers to the skilled person understands as to what upper limit the term may refer, if any.

The term “antibody” as used herein refers to any kind of immunoglobulin (Ig) or immunoglobulin derived peptide or protein that is capable of specifically binding to an epitope. Full length “antibodies” or “immunoglobulins” are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulphide bond, while the number of disulphide linkages varies between the heavy chain of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulphide bridges. Each heavy chain has an amino terminal variable domain (VH) followed by three carboxy terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Other forms of antibodies include heavy-chain antibodies, being those which consist only of two heavy chains and lack the two light chains usually found in antibodies. Heavy-chain antibodies include the hcIgG (IgG-like) antibodies of camelids such as dromedaries, camels, llamas and alpacas, and the IgNAR antibodies of cartilaginous fishes (for example sharks). And yet other forms of antibodies include single-domain antibodies (sdAb, called Nanobody by Ablynx, the developer) being an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies are typically produced from heavy-chain antibodies, but may also be derived from conventional antibodies. Antibodies (or those from which fragments thereof can be isolated) can include, for instance, chimeric, humanized, (fully) human, or hybrid antibodies with dual or multiple antigen or epitope specificities, antibody fragments and antibody sub-fragments, e.g., Fab, Fab' or F(ab')2 fragments, single chain antibodies (scFv) and the like, including hybrid fragments of any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions. Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. The light chains of human antibodies generally are classified as kappa and lambda light chains, and each of these contains one variable region and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Human IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. Human IgM subtypes include IgM, and IgM2. Human IgA subtypes include IgAl and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains ten or twelve heavy chains and ten or twelve light chains. Antibodies according to the invention may be IgG, IgE, IgD, IgA, or IgM immunoglobulins or fragments thereof.

A humanized antibody refers to immunoglobulin chains or fragments thereof (such as Fab, Fab', F(ab')2, Fv, or other antigen binding sub-sequences of antibodies), which contain minimal sequence (but typically, still at least a portion) derived from non- human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (the recipient antibody) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human species immunoglobulin (the donor antibody) such as a mouse, rat or rabbit having the desired specificity, affinity and capacity. As such, at least a portion of the framework sequence of said antibody or fragment thereof may be a human consensus framework sequence. In some instances, Fv framework residues of the human immunoglobulin need to be replaced by the corresponding non-human residues to increase specificity or affinity. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically at least two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which (e.g. human) immunoglobulin constant region may be modified (e.g. by mutations or glycol- engineering) to optimize one or more properties of such region and/or to improve the function of the (e.g. therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.

A chimeric antibody according to the invention refers to an antibody, whose light and/or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions which are identical to, or homologous to, corresponding sequences of different species, such as mouse and human. Alternatively, variable region genes derive from a particular antibody class or subclass while the remainder of the chain derives from another antibody class or subclass of the same or a different species. It covers also fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used for both the constant and variable domains.

Preferably, the term “antibody” in accordance with the present invention, refers to any immunoglobulin polypeptide derived from VDJ genomic sequences which comprises amino acid sequence stretches forming an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein.

Such an antibody may be, preferably, a monoclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies being still capable of binding to hypoglycosylated MUC1. Fragments and derivatives comprised by the term antibody as used herein encompass a bispecific antibody, a synthetic antibody, a Fab, F(ab)2, Fv or scFv fragment or a chemically modified derivative of any of these antibodies. Antibodies or fragments thereof, in general, can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals and, preferably, immunized mice.

An antibody of the present invention may also preferably be a polyclonal antibody. A polyclonal antibody according to the invention relates to a plurality of different antibody entities that are capable of recognizing the same or overlapping epitopes such that the polyclonal antibody is deemed to specifically bind to hypoglycosylated MUC1. The different antibody entities of a polyclonal antibody are typically found in the blood of immunized animals. It is well known how preparations such as sera comprising the polyclonal antibody can be provided.

The antibody of the present invention can be, preferably, generated by using the techniques described in the accompanying Examples below.

Preferably, the antibody of the invention shall specifically bind to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein.

The phrase “specifically binds to” as used in accordance with the present invention means that the antibody shall not cross-react significantly with components other than hyoglycosylated MUC1. Cross-reactivity of an antibody as mentioned herein can be tested by the skilled person by various techniques including immunological technologies such as Western blotting, ELISA or RIA based assays or measuring of binding affinities using, e.g., Biacore technology. Thus, the phrase “specifically binds to” as used herein means that the antibodies of the present invention make contact i) with a carbohydrate signature that is exposed on the hypoglycosylated MUC1, and ii) at the same time recognize the MUC-1 amino acid serine/threonine that the carbohydrate is attached to as well as neighboring MUC1 amino acids. Thus, the antibodies of the present invention recognize the carbohydrate moieties only in the specific MUC1 protein context, i.e. they do not bind to the carbohydrate moiety alone.

The term “epitope” as used herein is also known as antigenic determinant and refers to part of a substance, e.g. an immunogenic polypeptide, which is recognized by the immune system. Preferably, this recognition is mediated by the binding of antibodies, B cells, or T cells to the epitope in question. In this context, the term “binding” preferably relates to a specific binding. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The term “epitope” comprises both conformational and non-conformational epitopes. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In particular, the conformational epitopes are three dimensional epitopes whose architecture depends on the three dimensional structure of the surrounding amino acids in the native protein.

Preferably, the epitope to which the antibody binds is formed by a carbohydrate moiety of the hypoglycosylated MUC1 protein and by a peptide backbone portion of the MUC1 protein.

The term “carbohydrate moiety” refers to chemical derivatives of carbohydrates such as glycosides wherein the ligand includes a sugar group or pseudo-sugar group (such as pseudo sugar selected from a carba-sugar, an amino-sugar, an imino-sugar, an inositol, a polyhydroxylated piperidine, a pyrrolidine, a pyrrolizidine, an indolizidine and the like) attached to a non-sugar atom or molecule. A carbohydrate moiety as referred to herein consists, typically, of one or more monosaccharides. Preferably, the carbohydrate moiety the antibody of the present invention binds is an aberrant carbohydrate moiety. Preferably, the carbohydrate moiety” as used herein, refers to an aberrant carbohydrate moiety, i.e. carbohydrates with a qualitatively and/or quantitatively altered composition of monosaccharides compared to normal carbohydrates found on non-hypoglycosylated MUC1. Thus, an aberrant carbohydrate moiety may be composed of different monosaccharides compared to its counterpart found on MUC1 under physiological conditions and/or may be a truncated carbohydrate, i.e. a carbohydrate that consist of less monosaccharides compared to its counterpart found on MUC1 under physiological conditions. Various carbohydrates comprise the extensively branched O-glycan chains, such as N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylneuraminic acid (sialic acid, Neu5Ac) and fucose (Fuc). Such aberrations in the glycosylation typically occur during neoplastic transformation, wherein MUC1 is overexpressed and as a result of altered glycosyltransferase activity it undergoes incomplete glycosylation. The appearance of short truncated carbohydrate side chains, exposes MUC1 as a tumor antigen. Thus, the aberrant carbohydrate moiety to which the antibody of the present invention specifically binds to may be selected from the group consisting of Tn antigen (GalNAc), TF antigen (Gal(pi-3)GalNAc) and STn, sialyl Tn (Neu5Ac(a2-6)GalNAc) antigen (M. Movahedin et al., Glycobiology 2017), sialyl-Lewis^x antigen (Neu5Aca2-3Gaipi- 4[Fucal-3]GlcNAcP), sialyl-Lewis^a antigen (Neu5Aca2-3Gaipi-3[Fucal-4]GlcNAcP), and sialyl-di-Lewis^a.

Preferably, the carbohydrate moiety is sialyated N-acetylgalactosamine (GalNAc). The term “sialyated N-acetylgalactosamine” as used herein refers to an amino sugar derivative of galactose that has sialic acid as a terminal modification attacked via a glycosidic bond. The term “glycosidic bond” as used herein refers to type of ether bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate. Preferably, the carbohydrate moieties according to the present invention are attached to the peptide backbone via a nitrogen atom of the side-chain of asparagine, i.e. an N-linked glycosidic bond, or an oxygen atom of serine or threonine, i.e. an O-linked glycosidic bond. More preferably, the GalNAc shall be linked to a serine or threonine residue in the peptide backbone portion by a glycosidic bond. Preferably, the sialyated N-acetylgalactosamine (GalNAc) is linked to the serine or threonine residue in SEQ ID NO: 94 by a glycosidic bond.

In addition to the carbohydrate moiety of the hypoglycosylated MUC1 protein, the epitope of the antibody according to the present invention is also formed by a peptide backbone portion of the MUC1 protein. The phrase “peptide backbone portion of MUC1” as used herein, refers to a stretch of continuous amino acids in the MUC1 protein to which the antibody of the present invention specifically binds. Preferably, the peptide backbone portion refers to the MUC1 amino acid serine/threonine that the carbohydrate is attached to as well as neighboring MUC1 amino acids. More preferably, said peptide backbone portion of MUC1 comprises the amino acid sequence as shown in SEQ ID NO: 94 (RPAPGSTAP).

The term “MUC1” as used herein refers to the mucin- 1 protein encoded in humans by the MUC1 gene. It is a member of the mucin family and encodes a membrane bound, glycosylated phophoprotein. MUC1 serves a protective function by binding to pathogens and also acts in a cell signaling capacity. However, overexpression of MUC1 is often associated with cancer, such as colon, breast, ovarian, lung and pancreatic cancer. There are 17 isoforms described so far that are produced by alternative splicing and several orthologues of MUC1 have been reported in various animal species.

The MUC1 protein referred to in accordance with the present invention is, preferably, human MUC1 having an amino acid sequence as deposited under UniProt accession number Pl 5941. It will be understood that the term “MUC1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned MUC1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human MUC1 protein, preferably over the entire length of the said MUC1 proteins, respectively. The degree of identity between two amino acid sequences in accordance with the present invention can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm disclosed by Smith, by the homology alignment algorithm of Needleman, by the search for similarity method of Pearson, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologues. Variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologues.

Typically, MUC1 is extensively O-glycosylated and moderately N-glycosylated to yield mature functional mucin. In fact, glycosylation contributes to 50-90% of the total weight of MUC1 (Nath and Mukherjee, Trends Mol Med. 2014; 20(6): 332-342). However, cancer-associated MUC1 differs from MUC1 expressed in normal cells in that the protein is aberrantly glycosylated. In particular, cancer-associated MUC1 is often characterized with hypoglycosylation. Thus, the term “hypoglycosylated MUC1” as used herein refers to MUC1 proteins with reduced glycosylation compared to normal MUC1 proteins.

The antibody of the invention shall bind to hypoglycosylated MUC1, wherein the hypoglycosylated MUC1 is a cancer specific MUC1. Preferably, the MUC1 is specific to adenocarcinoma or sarcoma. More preferably, the cancer specific MUC1 is an adenocarcinoma specific MUC1.

The antibody of the invention is, preferably, obtainable by immunizing an animal with an immunogen. According to the present invention, immunizing an animal shall comprise a step of administering to the animal an immunogenic composition, typically, comprising a VAST- bound immunogen. The term “immunogen” in the context of the invention refers to any kind of compound or structure capable of inducing a specific immune response in a host when used in a vaccination or immunization procedure. Typically, the immunization composition is administered together with a pharmaceutically acceptable carrier and/or excipient, more preferably, such immunization composition comprises one or more adjuvants. An immunizing composition in accordance with the invention elicits an immune response in the immunized animal which is specific for hypoglycosylated MUC1, preferably, specific for an epitope by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein. Preferably, the immune response is the generation of antibodies against the immunization composition as described herein.

The term “VAST” refers to an immunogenic particle that comprises a plurality of VSGs on its surface, typically, in arrayed form, wherein to a significant portion of said VSGs the immunogen is bound. Typically, such a VSG-immunogen array can be generated by Sortase Tagging or other related techniques such as click chemistry. The immunogenic particle referred to above is, preferably, an inactivated trypanosome cell or membrane thereof comprising the VSGs as a result of natural or recombinant expression. The Trypanosome may be a recombinant Trypanosome that has been, e.g., UV inactivated. Details on Trypanosomal cells useful for the generation of VASTs are to be found elsewhere herein. Thus, the term “VAST-bound immunogen” refers to an immunogenic particle, comprising a UV inactivated trypanosome cell, wherein the trypanosome is an enzyme glycophosphatidylinositol phospholipace C (GPI-PLC)- negative trypanosome, wherein the immunogenic particle is coated with a plurality of variant surface glycoproteins that are covalently linked to an immunogen as described herein elsewhere. The term “free immunogen” refers to an immunogenic particle, wherein the immunogen as described herein elsewhere is covalently linked to variant surface glycoproteins of trypanosomes, however, the VSG-peptide is not associated with the cell membrane of the trypanosome in comparison with the VAST-bound immunogen.

The term “animal” as used herein refers to a non-human animal which is suitable for immunization and antibody production and from which antibody containing samples, such as blood, or splenic samples may be taken in order to isolate antibodies or antibody-producing cells, preferably, different types of B-cells. Accordingly, the animal shall have a humoral immune system. Suitable animals are birds, such as chicken, fish, such as sharks or mammals. Preferably, suitable animals are mammals, more preferably, laboratory animals such as rodents, most preferably, mice, or farming animals such as goat, sheep, pig or cow. Suitable animals may also include llamas and camels. Preferably, immunizing an animal comprises immunizing an animal with a VAST-bound immunogen, wherein said peptide VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein a sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Preferably, the antibody of the present invention is obtainable by immunizing an animal using an immunization scheme having the following steps: a) two times priming using VAST-bound immunogen; b) two times boosting using VAST-bound immunogen and free immunogen; and c) one time boosting using free immunogen.

The term “immunization scheme” as used herein refers to the schedule according to which the animal is immunized. Typically, immunization comprises more than one-time administration of the immunogen to the animal.

Preferably, the first administration of the immunogen to the animal is called “priming”. The said priming elicits, typically, a de novo immune response in the animal. Priming may sometimes require one or more separate administrations of the immunogen and is carried out until a memory of the immune system is established which result in a faster response of the immune system upon further challenges. Specifically, in accordance with the present invention, priming refers to the administration of a composition comprising a VAST-bound immunogen. Priming may be carried out at least once or at several time points, typically, two times with 4 weeks in between both priming immunizations.

The immunization scheme shall also typically comprise subsequent administrations of the immunogen to the animal called “boosting”. Boosting is sometimes referred to as an anamnestic response, i.e. an immune response in a previously sensitized subject and is intended to increase immunity against that immunogen back to protective levels after memory against that immunogen has declined through time. A boosting immunization can comprise multiple administrations of the immunogen, which may be the same or different amounts. Preferably, the boosting immunization is carried out by using a composition comprising free immunogen or a combination of VAST-bound immunogen and free immunogen. Boosting may be carried out once or at several time points, typically, two times with 4 weeks in between both boosting immunizations and 4 weeks between the last priming and the first boosting immunization. In accordance with the present invention, boosting, preferably, comprises two times boosting using VAST-bound immunogen and free immunogen followed by one time boosting using free immunogen. Preferably, immunogenic particles suitable as VASTs according to the present invention have been described in W02020/084072 or WO2021/214043.

More preferably, the VASTs according to the present invention may be generated by a method comprising the steps of:

A) providing recombinant Trypanosoma brucei cells expressing a VSG, preferably, a sortaggable VSG;

B) sortagging the cells of step a) with an immunogen as described herein elsewhere;

C) UV irradiating the sortagged cells of steb b) until inactivation;

D) washing the UV inactivated cells to obtain a membrane suspension; and

E) identifying VASTs from the membrane suspension obtained in step d) by microscopy and flow cytometry

Steps B) and C) may be carried out in opposite order as well.

The method for generating the VASTs may consist of the aforementioned steps or may comprise further steps, such as steps for producing recombinant Trypanosoma brucei cells expressing, preferably, sortaggable VSG or steps aiming at sortagging or coupling desired compounds to the VSG by sortase or chemical reactions. Moreover, there might be additional steps within or between the steps recited above.

In step A) of the method for generating the VASTs, recombinant Trypanosoma brucei cells expressing, preferably, sortaggable VSG are provided.

The term “recombinant Trypanosoma brucei cells” refers to a single cell parasitic organism. Trypanosoma brucei is a pathogenic organism responsible for the sleeping sickness. Trypanosoma brucei as referred to herein encompasses all subspecies such as Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Trypanosoma brucei cells can be genetically modified and, thus, allow for the generation of recombinant organisms. Trypanosoma brucei exhibits on its surface glycoprotein coat comprising the variant surface glycoprotein (VSG).

The term “VSG” as used herein refers to a glycoprotein belonging into a class of glycoproteins of approximately 60-kDa as a monomer which may be found as monomers, dimers, trimers or multimers densely located on the outer membrane in order to form a surface coat. VSG dimers make up approx. 90% of all cell surface proteins in trypanosomes. VSGs are highly immunogenic and an immune response raised against a specific VSG coat will rapidly kill trypanosomes expressing this variant. However, with each cell division there is a chance that the progeny will switch expression of a VSG gene to another to change the VSG that is being expressed and, thus, escape immunity. Only one VSG gene is expressed at a time. VSG expression is 'switched' by homologous recombination induced by double-strand breaks of a silent basic copy gene from an array (directed by homology) into the active telomerically- located expression site. VSG annotation, protein sequences and gene sequences are derivable from public databases such as www.ensemble.org. A collection of VSGs of Trypanosoma brucei brucei (Lister 427 strain) is published in Cross 2014, Mol. Biochem. Parasitol. 195(l):59-73. Preferred VSGs in context of the present invention are Trypanosoma, brucei VSGs, more preferably VSG2, VSG3, ILTatl.24, VSG11, VSG13, VSGsur, VSG1954, or VSG531. A preferred VSG according to the invention which is characterized by an N-terminus which is located 3 -dimensionally within the VSG at a position which is, when the VSG is present within a VSG coat, for example, on a trypanosome cell, located sufficiently close to the accessible outer coat-surface such that the addition of a linker sequence, preferably not more than 100 amino acids, preferably not more than 50 amino acids, and most preferably not more than 20 amino acids in length, allows for modification of the so extended or not extended N- terminus of the VSG protein. Preferably, the insertion may be immediately downstream of the signal peptide cleavage site.

Moreover, the recombinant Trypanosoma brucei cells, preferably, lack GPI phospholipase C. The genetic deletion of the endogenous glycophosphatidylinositol phospholipase C (GPI-PLC), i.e. the enzyme that "sheds" VSG off the surface of dying cells, is crucial to generating T. brucei that can be used, e.g., as antigen display platform, because unless GPI-PLC is removed from the genome, any form of inactivation of the trypanosome cells will also lead to the disintegration of the VSG coat and of the cell itself. Once VSGs are shed due to the action of GPI-PLC, the VSG coat disintegrates and the cells lyse.

In step B) of the method for generating the VASTs, the cells of step a) are sortagged with an immunogen as described herein elsewhere. The term “sortaggable” as used herein refers to a VSG that can be linked to another polypeptide or peptide by sortase activity. Preferably, the VSG will comprise a sortase donor or a sortase acceptor amino acid sequence. Typically, said sortase donor or acceptor sequence may be introduced into the VSG by genetic engineering. For example, the said sortase donor or acceptor sequence may be comprised in a peptide that will be fused to the VSG. Alternatively, a suitable endogenous sequence of the VSG may be genetically modified such that it becomes a sortase donor or acceptor sequence.

A sortase as used herein, refers to a protein having sortase activity, i.e., an enzyme able to carry out a transpeptidation reaction conjugating the C-terminus of a protein to the N-terminus of a protein via transamidation. The term includes full-length sortase proteins, e.g., full-length naturally-occurring sortase proteins, fragments of such sortase proteins that have sortase activity, modified (e.g., mutated) variants or derivatives of such sortase proteins or fragments thereof, as well as proteins that are not derived from a naturally occurring sortase protein, but exhibit sortase activity. Those of skill in the art will readily be able to determine whether or not a given protein or protein fragment exhibits sortase activity, e.g., by contacting the protein or protein fragment in question with a suitable sortase substrate under conditions allowing transpeptidation and determining whether the respective transpeptidation reaction product is formed. Suitable sortases will be apparent to those of skill in the art and include, but are not limited to, sortase A, sortase B, sortase C, and sortase D type sortases. Suitable sortases are described, for example, in Dramsi 2005, Res. Microbiol. 156(3):289-97, Comfort 2004, Infect Immun, 72(5):2710-22, Chen 2011, Proc Natl Acad. Sci. USA. Jul. 12; 108(28): 11399, and Pallen 2001, Trends in Microbiology, 2001, 9(3), 97-101. Moreover, the present invention encompasses embodiments relating to a sortase A from any bacterial species or strain. Those of skill in the art will appreciate that any sortase and any sortase recognition motif can be used in some embodiments of this invention, including, but not limited to, the sortases and sortase recognition motifs described in WO2010/087994, WO2011/133704, and WO 2020/84072.

The sortase substrates are amino acid sequences that can be utilized in a sortase-mediated transpeptidation reaction. Typically, a sortase utilizes two substrates, a substrate comprising a C -terminal sortase recognition motif, and a second substrate comprising an N-terminal sortase recognition motif and the transpeptidation reaction results in a conjugation of both substrates via a covalent bond. In context of the invention the “C-terminal sortase recognition motif’ is also referred to as “sortagging donor sequence”, whereas the term “N-terminal sortase recognition motif’ is referred to as “sortagging acceptor sequence”. Preferably, the C-terminal and N-terminal recognition motifs are comprised in different amino acid sequences, for example, one N-terminally of the VSG, and the other linked to the immunogen such that there is a free carboxyl group at the end of the sortagging donor site. Some sortase recognition motifs are described herein and additional suitable sortase recognition motifs are well-known to those of skill in the art. Sortase recognition motifs will be apparent to those of skill in the art. A sortase substrate may comprise additional moieties or entities apart from the peptidic sortase recognition motif.

For example, a sortase substrate may comprise an LPXTG/A motif (SEQ ID Nos: 98 and 99), the N-terminus of which is conjugated to any agent, (e.g. a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label). Similarly, a sortase substrate may comprise an oligoglycine (Gl-5) motif or oligoalanine motif, preferably G3 or G5, the C- terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label. Accordingly, sortase substrates are not limited to proteins or peptides but include any moiety or entity conjugated to a sortase recognition motif.

The VSG shall be, preferably, “sortaggable”, i.e. it shall be a sortase substrate and, thus, comprise a sortase donor sequence or sortase acceptor sequence. An example of a sortaggable VSG can be derived from Pinger 2017, Nat Commun. 8(1): 828. Depending on whether the VSG comprises a sortase acceptor or donor, a molecule to be linked by sortagging to the VSG shall comprise the complement, i.e. in case of the sortase acceptor being comprised by the VSG, it shall comprise a sortase donor or in case of a sortase donor being comprised by the VSG, it shall comprise a sortase acceptor.

It will be understood that said VSGs may also be modified chemically. Preferably, said VSGs may be modified by chemical coupling reactions in order to couple desired molecules such as targeting compounds specified elsewhere herein. Typically, targeting compound may be covalently linked, preferably via a linker, to the N-terminus of the VSG. Targeting compounds may be linked to the VSG by any techniques known in the art, including any chemical reaction. Preferably, click-chemistry or other cross-linking may be used. Click-chemistry in context of the invention shall refer to chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. Any variants of such approaches may be used to connect the targeting compound to the VSG in accordance with the invention. In general, the linking of the VSG to the targeting compound may include the use of any linking means or linker, which in context of the herein disclosed invention refers to the means by which the VSG and the targeting compound are linked or connected to form a modified VSG. The one or more linkers or linking means for linking the VSG and the targeting compound may be any structurally suitable means to connect the two. Exemplary linkers include the use of one or more amino acids which may be used to form a peptide, in some embodiments having a modified peptide backbone, a small chemical scaffold, a biotin-streptavidin, an organic or inorganic nanoparticle, a polynucleotide sequence, peptide-nucleic acids, an organic polymer, or an immunoglobulin Fc domain. The means for linking can comprise covalent and/or noncovalent bonds. The one or more linkers can include various sequences or other structural features that provide various functions or properties. For example, the one or more linkers can contain structural elements to allow the VSG to be derivatized.

Preferably, the recombinant Trypanosoma brucei cells expressing, preferably, sortaggable VSG are provided in purified or partially purified form. Typically, the said cells shall be free or essentially free of any cultivation media. Preferably, such a preparation of recombinant Trypanosoma brucei cells expressing, preferably, sortaggable VSG can obtained by centrifugation allowing for separation of media and cells and resuspension of the cells in a suitable solvent such as the isotonic solution to be used for carrying out the subsequent step b). In case that preparation of recombinant Trypanosoma bruce\ cells expressing sortaggable VSG shall be stored, an isotonic solution which allows for storage of the cells may be used. Suitable solutions are well-known to the person skilled in the art. Particular preferred techniques for providing the recombinant T. brucei cells are described in the accompanying Examples, below. These techniques may encompass centrifugation of the cells in order to obtain a pellet of cells as well carrying out one or more washing steps.

In step C) of the method for generating the VASTs, the sortagged cells of step b) are inactivated. Preferably, the inactivation of the trypanosome cells is achieved by UV irradiation in irradiation buffer. The irradiation buffer may be, for example, phosphate-buffered saline (PBS) supplemented with glucose or HMI-9 medium without serum. The UV irradiation step may also be performed prior to sortagging of the cells.

In step D) of the method of generating VASTs, a membrane suspension is obtained by washing and resuspending the UV inactivated cells of step C) in the same buffer used for UV irradiation.

In step E) of the method of the invention, the VASTs are identified from the membrane suspension obtained in step d) by fluorescence microscopy or flow cytometry analysis. Fluorescence microscopy and flow cytometry are well known and widely described methods. For example, a fluorescent moiety, e.g. a fluorophoe like 6-FAM, can be attached to the N- terminus of the sortase donor sequence as described herein elsewhere. By using specific monoclonal antibodies that bind to 6-FAM, efficiency of sortagging can be determined or VASTs can be identified from the membrane suspension by direct FACS analysis or fluorescence microscopy.

Yet preferably, the antibody according to the present invention may be a monoclonal antibody. The said monoclonal antibody of the invention in addition to the above-mentioned functional features shall, preferably, be characterized by at least one of the following structural features.

The monoclonal antibody, preferably, comprises a pair of light and heavy chains havin an amino acid sequence as indicated by SEQ ID NOs in Table 1. The amino acid sequence of the light and/or heay chain in the monoclonal antibody according to the present invention may be a variant sequence and, thus differ by at least one amino acid sequence substitution, addition and/or deletion from those shown in the SEQ ID NOs. Preferably, the said variant sequence is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific amino acid sequence shown in the SEQ ID NO. It will be understood that such variant sequences of light and heavy chains defined by SEQ ID NOs referred to hererin shall contain the same CDR1, CDR2 and CDR3 sequences as the light or heavy chain defined by SEQ ID NOs. Accordingly, the sequence variation shall not occur within the CDR sequences. Moreover, the said variant sequences of light and heavy chains referred to hererin shall contain the same CDR1, CDR2 and CDR3 and FR1, FR2, FR3 and FR4 sequences as the light or heavy chain defined by SEQ ID NOs. Accordingly, the sequence variation shall not occur within the CDR and FR sequences. In the following, this is meant by, e. g., a heavy chain as defined in (al) or (bl) and wherein said heavy chain is at least 80% identical to SEQ ID NO: 23, and a light chain as defined in (al) or (bl) and wherein said light chain is at least 80% identical to SEQ ID NO: 1.

Sequence identity as referred to in accordance with the present invention can be determined by comparing two sequences with each other such that the number of identical amino acids between the sequences can be identified. Sequence identity can be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID, if not specifically indicated otherwise. The sequence identity can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul, with the well-known CLUSTAL algorithm, the BLAST, BLAT or BlastZ (or BlastX) algorithms using default parameters.

Preferably, the monoclonal antibody of the invention is characterized in that it comprises a light chain having the CDR1, CDR2 and CDR3 as indicated by the respective SEQ ID NO in the Table 1 and a heavy chain having the CDR1, CDR2 and CDR3 as indicated by the respective SEQ ID NO in the Table 1 wherein said light chain and heavy chain CDRs are combinations as indicated in the Table 1.

able 1: Monoclonal antibodies (mAbs)

Particular preferred antibodies according to the present invention comprise a combination of a heavy chain and a light chain selected from the group of combinations consisting of:

• SEQ ID Nos: 1 and 23 (mAb 1061871);

• SEQ ID Nos: 5 and 27 (mAb 1061917);

• SEQ ID Nos: 7 and 31 (mAb 1062027);

• SEQ ID Nos: 8 and 35 (mAb 1062313);

• SEQ ID Nos: 12 and 38 (mAb 1062919);

• SEQ ID Nos: 15 and 42 (mAb 1062845);

• SEQ ID Nos: 19 and 46 (mAb 1062853);

• SEQ ID Nos: 100 and 148 (mAb 47);

• SEQ ID Nos: 100 and 152 (mAb 207);

• SEQ ID Nos: 100 and 154 (mAb 53);

• SEQ ID Nos: 100 and 157(mAb 599);

• SEQ ID Nos: 104 and 161 (mAb 101);

• SEQ ID Nos: 104 and 165 (mAb 105);

• SEQ ID Nos: 105 and 167 (mAb 135);

• SEQ ID Nos: 109 and 282 (mAb 149);

• SEQ ID Nos: 109 and 171 (mAb 261);

• SEQ ID Nos: 113 and 173 (mAb 815);

• SEQ ID Nos: 117 and 176 (mAb 971);

• SEQ ID Nos: 119 and 179 (mAb 205);

• SEQ ID Nos: 123 and 183 (mAb 247);

• SEQ ID Nos: 127 and 185 (mAb 407);

• SEQ ID Nos: 127 and 189 (mAb 415);

• SEQ ID Nos: 131 and 195 (mAb 493);

• SEQ ID Nos: 131 and 195 (mAb 659);

• SEQ ID Nos: 135 and 197 (mAb 961);

• SEQ ID Nos: 138 and 201 (mAb 691);

• SEQ ID Nos: 141 and 201 (mAb 359); and

• SEQ ID Nos: 144 and 205 (mAb 767).

Most preferably, antibodies according to the present invention comprise the combination of SEQ ID Nos: 19 and 46 or SEQ ID Nos: 8 and 35. The combination of SEQ ID Nos: 19 and 46 were present in 2 separate B cells isolated after immunization, suggesting that this antibody has been expanded within the immunized mice. SEQ ID Nos: 8 and 35 were isolated from an IgG class switched B cell with the highest amount of observed somatic hypermutations, suggestive of immunization-dependent affinity maturation. Depending on the antibody type envisaged, the antibody of the invention may further comprise amino acids or amino acid sequence from the framework regions. The term "framework regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. From N-terminal to C-terminal, light chain variable region and heavy chain variable region both typically have the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Table 2: Framework regions of monoclonal antibodies

Advantageously, it has been found in accordance with the studies underlying the present invention that the antibodies described herein can differentiate between normal carbohydrates and aberrant carbohydrates on cancer cells. Due to their binding profile, i.e. they recognize a carbohydrate signature that is exposed on hypoglycosylated MUC1 and at the same time recognize the MUC1 amino acid serine/threonine that the carbohydrate is attached to as well as neighboring MUC1 amino acids, the antibodies are highly specific for cancer cells and thus, have a very low risk for unspecific off-target binding. This binding profile also ensures that the antibodies only target cells that express hypoglycosylated MUC1, while healthy cells exposing carbohydrates in e.g. inflammatory context will not be targeted. Surprisingly, the antibodies of the present invention have a deep binding pocket maximizing the contact are of the antibody to its target. As a result, this special mode of binding leads to a highly improved affinity. Using the specific imunogens and the immunization scheme described elsewhere herein was surprisingly found to allow for generating the antibody of the invention. The immunogen of the invention is a VAST-bound immunogen, wherein said immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92, wherein a sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond. Using said immunogen, antibodies that specifically bind to hypoglycosylated MUC1, wherein said antibodies bind to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein could be efficiently generated.

The present invention also relates to an antibody as defined herein for use in treating and/or preventing cancer associated with aberrant cancer specific MUC1 expression. Preferably, the cancer is an adenocarcinoma or sarcoma.

The term "treating" as used herein refers to any improvement, cure or amelioration of the disease or condition as referred to herein. Preferably, the disease or condition is cancer associated with aberrant cancer specific MUC1 expression. Cancer types associated with aberrant cancer specific MUC1 expression shall, preferably, be epithelial cancers, more preferably, selected from the group consisting of epithelial cancer types, such as lung, liver, colon, ovarian, pancreatic, bladder, glial, kidney, uterine, intestinal and breast cancer. Preferably, the cancer is an adenocarcinoma or sarcoma. It will be understood that treatment may not occur in 100% of the subjects to which the antibody has been administered. The term, however, requires that the treatment occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-te st, Mann- Whitney-U test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.05, 0.01, 0.005, 0.001, or 0.0001.

The term “preventing” as used herein refers to significantly reducing the likelihood with which the disease or condition develops in a subject within a defined window (prevention window) starting from the administration of the antibody onwards. Typically, the prevention window is a time window starting from the onset of the therapy from 1 to 5 months or from 1 to 3 years or from 3 to 5 years. The prevention window depends on the amount of antibody which is administered and the applied dosage regimen. Typically, suitable prevention windows can be determined by the clinician based on the amount of antibody to be administered and the dosage regimen to be applied without further ado. It will be understood that prevention may not occur in 100% of the subjects to which the antibody has been administered. The term, however, requires that the prevention occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney-U test etc. Details are described elsewhere herein.

The antibody for use in accordance with the present invention may be, preferably, formulated as a pharmaceutical composition. By way of example, the pharmaceutical composition of the invention may comprise between 0.1% and 100% (w/w) active ingredient, such as about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably between about 1% and about 20%, between about 10% and 50% or between about 40% and 90%. The pharmaceutical composition according to the invention may comprise further ingredients such as carriers, stabilizers and/or solvents. As used herein, such carriers, stabilizers and/or solvents include any and all solvents, solubilisers, fillers, stabilisers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption-delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions. The pharmaceutical composition is, typically, formulated to be compatible with its intended route of administration. Examples of routes of administration include oral, parenteral, e.g., intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, intraperitoneal, transdermal (topical) and transmucosal administration.

The present invention also refers to an antibody as defined as follows:

(al) a heavy chain having a CDR1 as shown in SEQ ID NO: 24, a CDR2 as shown in SEQ ID NO: 25, a CDR3 as shown in SEQ ID NO 26, and a light chain having a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO 4;

(bl) a heavy chain having a FR1 as shown in SEQ ID NO: 69, a CDR1 as shown in SEQ ID NO: 24, an FR2 as shown in SEQ ID NO: 70, a CDR2 as shown in SEQ ID NO: 25, an FR3 as shown in SEQ ID NO: 71, a CDR3 as shown in SEQ ID NO: 26 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 2, an FR2 as shown in SEQ ID NO: 51, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(cl) a heavy chain as shown in SEQ ID NO: 23 and a light chain shown in SEQ ID NO: 1; or

(dl) a heavy chain as defined in (al) or (bl) and wherein said heavy chain is at least 80% identical to SEQ ID NO: 23, and a light chain as defined in (al) or (bl) and wherein said light chain is at least 80% identical to SEQ ID NO: 1; or

(a2) a heavy chain having a CDR1 as shown in SEQ ID NO: 28, a CDR2 as shown in SEQ ID NO: 29, a CDR3 as shown in SEQ ID NO: 30, and a light chain having a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO 6;

(b2) a heavy chain having a FR1 as shown in SEQ ID NO: 73, a CDR1 as shown in SEQ ID NO: 28, an FR2 as shown in SEQ ID NO: 74, a CDR2 as shown in SEQ ID NO: 29, an FR3 as shown in SEQ ID NO: 75, a CDR3 as shown in SEQ ID NO: 30 and an FR4 as shown in SEQ ID NO: 76, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 2, an FR2 as shown in SEQ ID NO: 51, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(c2) a heavy chain as shown in SEQ ID NO: 27 and a light chain shown in SEQ ID NO: 5; or

(d2) a heavy chain as defined in (al) or (bl), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 27, and a light chain as defined in (a2) or (b2) and wherein said light chain is at least 80% identical to SEQ ID NO: 5; or

(a3) a heavy chain having a CDR1 as shown in SEQ ID NO: 32, a CDR2 as shown in SEQ ID NO: 33, a CDR3 as shown in SEQ ID NO: 34, and a light chain having a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO 4;

(b3) a heavy chain having a FR1 as shown in SEQ ID NO: 77, a CDR1 as shown in SEQ ID NO: 32, an FR2 as shown in SEQ ID NO: 78, a CDR2 as shown in SEQ ID NO: 33, an FR3 as shown in SEQ ID NO: 79, a CDR3 as shown in SEQ ID NO: 34 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 2, an FR2 as shown in SEQ ID NO: 51, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(c3) a heavy chain as shown in SEQ ID NO: 31 and a light chain shown in SEQ ID NO: 7; or (d3) a heavy chain as defined in (a3) or (b3), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 31, and a light chain as defined in (a3) or (b3) and wherein said light chain is at least 80% identical to SEQ ID NO: 7; or

(a4) a heavy chain having a CDR1 as shown in SEQ ID NO: 36, a CDR2 as shown in SEQ ID NO: 29, a CDR3 as shown in SEQ ID NO: 37, and a light chain having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10, a CDR3 as shown in SEQ ID NO 11; (b4) a heavy chain having a FR1 as shown in SEQ ID NO: 80, a CDR1 as shown in SEQ ID NO: 36, an FR2 as shown in SEQ ID NO: 74, a CDR2 as shown in SEQ ID NO: 29, an FR3 as shown in SEQ ID NO: 81, a CDR3 as shown in SEQ ID NO: 37 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 54, a CDR1 as shown in SEQ ID NO: 9, an FR2 as shown in SEQ ID NO: 55, a CDR2 as shown in SEQ ID NO: 10, an FR3 as shown in SEQ ID NO: 56, a CDR3 as shown in SEQ ID NO: 11 and an FR4 as shown in SEQ ID NO: 57;

(c4) a heavy chain as shown in SEQ ID NO: 35 and a light chain shown in SEQ ID NO: 8; or

(d4) a heavy chain as defined in (a4) or (b4), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 35, and a light chain as defined in (a4) or (b4) and wherein said light chain is at least 80% identical to SEQ ID NO: 8; or

(a5) a heavy chain having a CDR1 as shown in SEQ ID NO: 39, a CDR2 as shown in SEQ ID NO: 40, a CDR3 as shown in SEQ ID NO: 41, and a light chain having a CDR1 as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 10, a CDR3 as shown in SEQ ID NO: 12;

(b5) a heavy chain having a FR1 as shown in SEQ ID NO: 83, a CDR1 as shown in SEQ ID NO: 39, an FR2 as shown in SEQ ID NO: 84, a CDR2 as shown in SEQ ID NO: 40, an FR3 as shown in SEQ ID NO: 85, a CDR3 as shown in SEQ ID NO: 41 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 58, a CDR1 as shown in SEQ ID NO:, an FR2 as shown in SEQ ID NO: 59, a CDR2 as shown in SEQ ID NO:, an FR3 as shown in SEQ ID NO: 60, a CDR3 as shown in SEQ ID NO: and an FR4 as shown in SEQ ID NO: 61;

(c5) a heavy chain as shown in SEQ ID NO: 38 and a light chain shown in SEQ ID NO: 12; or

(d5) a heavy chain as defined in (a5) or (b5), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 38, and a light chain as defined in (a5) or (b5) and wherein said light chain is at least 80% identical to SEQ ID NO: 12; or (a6) a heavy chain having a CDR1 as shown in SEQ ID NO: 43, a CDR2 as shown in SEQ ID NO: 44, a CDR3 as shown in SEQ ID NO: 45, and a light chain having a CDR1 as shown in SEQ ID NO: 16, a CDR2 as shown in SEQ ID NO: 17, a CDR3 as shown in SEQ ID NO: 18;

(b6) a heavy chain having a FR1 as shown in SEQ ID NO: 86, a CDR1 as shown in SEQ ID NO: 43, an FR2 as shown in SEQ ID NO: 87, a CDR2 as shown in SEQ ID NO: 44, an FR3 as shown in SEQ ID NO: 88, a CDR3 as shown in SEQ ID NO: 45 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 62, a CDR1 as shown in SEQ ID NO: 16, an FR2 as shown in SEQ ID NO: 63, a CDR2 as shown in SEQ ID NO: 17, an FR3 as shown in SEQ ID NO: 64, a CDR3 as shown in SEQ ID NO: 18 and an FR4 as shown in SEQ ID NO: 65;

(c6) a heavy chain as shown in SEQ ID NO: 42 and a light chain shown in SEQ ID NO: 15; or

(d6) a heavy chain as defined in (a6) or (b6), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 42, and a light chain as defined in (a6) or (b6) and wherein said light chain is at least 80% identical to SEQ ID NO: 15; or

(a7) a heavy chain having a CDR1 as shown in SEQ ID NO: 47, a CDR2 as shown in SEQ ID NO: 48, a CDR3 as shown in SEQ ID NO: 49, and a light chain having a CDR1 as shown in SEQ ID NO: 20, a CDR2 as shown in SEQ ID NO: 21, a CDR3 as shown in SEQ ID NO: 22;

(b7) a heavy chain having a FR1 as shown in SEQ ID NO: 89, a CDR1 as shown in SEQ ID NO: 47, an FR2 as shown in SEQ ID NO: 90, a CDR2 as shown in SEQ ID NO: 48, an FR3 as shown in SEQ ID NO: 91, a CDR3 as shown in SEQ ID NO: 49 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 66, a CDR1 as shown in SEQ ID NO: 20, an FR2 as shown in SEQ ID NO: 67, a CDR2 as shown in SEQ ID NO: 21, an FR3 as shown in SEQ ID NO: 68 a CDR3 as shown in SEQ ID NO: 22 and an FR4 as shown in SEQ ID NO: 61;

(c7) a heavy chain as shown in SEQ ID NO: 46 and a light chain shown in SEQ ID NO: 19; or

(d7) a heavy chain as defined in (a7) or (b7), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 46, and a light chain as defined in (a7) or (b7) and wherein said light chain is at least 80% identical to SEQ ID NO: 19; or

(a8) a heavy chain having a CDR1 as shown in SEQ ID NO: 149, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 151, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 102, a CDR3 as shown in SEQ ID NO: 103; (b8) a heavy chain having a FR1 as shown in SEQ ID NO: 242, a CDR1 as shown in SEQ ID NO: 149, an FR2 as shown in SEQ ID NO: 243, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 151 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 209, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 102, an FR3 as shown in SEQ ID NO: 211 a CDR3 as shown in SEQ ID NO: 103 and an FR4 as shown in SEQ ID NO: 212;

(c8) a heavy chain as shown in SEQ ID NO: 148 and a light chain shown in SEQ ID NO: 100; or

(d8) a heavy chain as defined in (a8) or (b8), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 148, or, and a light chain as defined in (a8) or (b8) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(a9) a heavy chain having a CDR1 as shown in SEQ ID NO: 149, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 153, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 102, a CDR3 as shown in SEQ ID NO: 103;

(b9) a heavy chain having a FR1 as shown in SEQ ID NO: 242, a CDR1 as shown in SEQ ID NO: 149, an FR2 as shown in SEQ ID NO: 243, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 153 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 209, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 102, an FR3 as shown in SEQ ID NO: 211, a CDR3 as shown in SEQ ID NO: 103 and an FR4 as shown in SEQ ID NO: 212;

(c9) a heavy chain as shown in SEQ ID NO: 152 and a light chain shown in SEQ ID NO: 100; or

(d9) a heavy chain as defined in (a9) or (b9), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 152, and a light chain as defined in (a9) or (b9) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(alO) a heavy chain having a CDR1 as shown in SEQ ID NO: 155, a CDR2 as shown in SEQ ID NO: 33, a CDR3 as shown in SEQ ID NO: 156, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 102, a CDR3 as shown in SEQ ID NO: 103;

(blO) a heavy chain having a FR1 as shown in SEQ ID NO: 77, a CDR1 as shown in SEQ ID NO: 155, an FR2 as shown in SEQ ID NO: 245, a CDR2 as shown in SEQ ID NO: 33, an FR3 as shown in SEQ ID NO: 79, a CDR3 as shown in SEQ ID NO: 156 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 209, a CDR1 as shown in SEQ ID NO: 209, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 102, an FR3 as shown in SEQ ID NO: 211 a CDR3 as shown in SEQ ID NO: 103 and an FR4 as shown in SEQ ID NO: 212;

(clO) a heavy chain as shown in SEQ ID NO: 154 and a light chain shown in SEQ ID NO: 100; or

(dlO) a heavy chain as defined in (alO) or (blO), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 154, and a light chain as defined in (alO) or (blO) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(al 1) a heavy chain having a CDR1 as shown in SEQ ID NO: 158, a CDR2 as shown in SEQ ID NO: 159, a CDR3 as shown in SEQ ID NO: 160, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 102, a CDR3 as shown in SEQ ID NO: 103;

(bl 1) a heavy chain having a FR1 as shown in SEQ ID NO: 246, a CDR1 as shown in SEQ ID NO: 158, an FR2 as shown in SEQ ID NO: 247, a CDR2 as shown in SEQ ID NO: 159, an FR3 as shown in SEQ ID NO: 248, a CDR3 as shown in SEQ ID NO: 160 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 209, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 102, an FR3 as shown in SEQ ID NO: 211 a CDR3 as shown in SEQ ID NO: 103 and an FR4 as shown in SEQ ID NO: 212;

(cl 1) a heavy chain as shown in SEQ ID NO: 157 and a light chain shown in SEQ ID NO: 100; or

(dl 1) a heavy chain as defined in (al 1) or (bl 1), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 157, and a light chain as defined in (al l) or (bl 1) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(al2) a heavy chain having a CDR1 as shown in SEQ ID NO: 162, a CDR2 as shown in SEQ ID NO: 163, a CDR3 as shown in SEQ ID NO: 164, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO: 4; (bl 2) a heavy chain having a FR1 as shown in SEQ ID NO: 249, a CDR1 as shown in SEQ ID NO: 162, an FR2 as shown in SEQ ID NO: 250, a CDR2 as shown in SEQ ID NO: 163, an FR3 as shown in SEQ ID NO: 251, a CDR3 as shown in SEQ ID NO: 164 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(cl2) a heavy chain as shown in SEQ ID NO: 161 and a light chain shown in SEQ ID NO: 104; or (dl2) a heavy chain as defined in (al2) or (bl2), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 161, and a light chain as defined in (al2) or (bl2) and wherein said light chain is at least 80% identical to SEQ ID NO: 104; or

(al3) a heavy chain having a CDR1 as shown in SEQ ID NO: 158 , a CDR2 as shown in SEQ ID NO: 159, a CDR3 as shown in SEQ ID NO: 160, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO: 4; (bl 3) a heavy chain having a FR1 as shown in SEQ ID NO: 249, a CDR1 as shown in SEQ ID NO: 158, an FR2 as shown in SEQ ID NO: 250, a CDR2 as shown in SEQ ID NO: 159, an FR3 as shown in SEQ ID NO: 251, a CDR3 as shown in SEQ ID NO: 160 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(cl3) a heavy chain as shown in SEQ ID NO: 165 and a light chain shown in SEQ ID NO: 104; or

(dl 3) a heavy chain as defined in (al3) or (b 13), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 165, and a light chain as defined in (al3) or (b 13) and wherein said light chain is at least 80% identical to SEQ ID NO: 104; or

(al4) a heavy chain having a CDR1 as shown in SEQ ID NO: 168, a CDR2 as shown in SEQ ID NO: 169, a CDR3 as shown in SEQ ID NO: 170, and a light chain having a CDR1 as shown in SEQ ID NO: 106, a CDR2 as shown in SEQ ID NO: 107, a CDR3 as shown in SEQ ID NO: 108;

(bl 4) a heavy chain having a FR1 as shown in SEQ ID NO: 252, a CDR1 as shown in SEQ ID NO: 168, an FR2 as shown in SEQ ID NO: 253, a CDR2 as shown in SEQ ID NO: 169, an FR3 as shown in SEQ ID NO: 254, a CDR3 as shown in SEQ ID NO: 170 and an FR4 as shown in SEQ ID NO: 255, and a light chain having an FR1 as shown in SEQ ID NO: 213, a CDR1 as shown in SEQ ID NO: 106, an FR2 as shown in SEQ ID NO: 214, a CDR2 as shown in SEQ ID NO: 107, an FR3 as shown in SEQ ID NO: 215, a CDR3 as shown in SEQ ID NO: 108 and an FR4 as shown in SEQ ID NO: 61;

(cl4) a heavy chain as shown in SEQ ID NO: 167 and a light chain shown in SEQ ID NO: 105; or

(dl4) a heavy chain as defined in (al4) or (bl4), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 167, and a light chain as defined in (al4) or (bl4) and wherein said light chain is at least 80% identical to SEQ ID NO: 105; or (al 5) a heavy chain having a CDR1 as shown in SEQ ID NO: 283, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 284, and a light chain having a CDR1 as shown in SEQ ID NO: 110, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 112;

(bl 5) a heavy chain having a FR1 as shown in SEQ ID NO: 256, a CDR1 as shown in SEQ ID NO: 283, an FR2 as shown in SEQ ID NO: 257, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 284 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 216, a CDR1 as shown in SEQ ID NO: 110, an FR2 as shown in SEQ ID NO: 217, a CDR2 as shown in SEQ ID NO: 111, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 112 and an FR4 as shown in SEQ ID NO: 61;

(cl 5) a heavy chain as shown in SEQ ID NO: 282 and a light chain shown in SEQ ID NO: 109; or

(dl 5) a heavy chain as defined in (al 5) or (bl 5), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 282, and a light chain as defined in (al5) or (b 15) and wherein said light chain is at least 80% identical to SEQ ID NO: 109; or

(al6) a heavy chain having a CDR1 as shown in SEQ ID NO: 283, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 172, and a light chain having a CDR1 as shown in SEQ ID NO: 110, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 112;

(bl 6) a heavy chain having a FR1 as shown in SEQ ID NO: 256, a CDR1 as shown in SEQ ID NO: 283, an FR2 as shown in SEQ ID NO: 257, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 172 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 216, a CDR1 as shown in SEQ ID NO: 110, an FR2 as shown in SEQ ID NO: 217, a CDR2 as shown in SEQ ID NO: 111, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 112 and an FR4 as shown in SEQ ID NO: 61;

(cl6) a heavy chain as shown in SEQ ID NO: 171 and a light chain shown in SEQ ID NO: 109; or

(dl6) a heavy chain as defined in (al6) or (bl6), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 171, and a light chain as defined in (al6) or (bl6) and wherein said light chain is at least 80% identical to SEQ ID NO: 109; or

(al7) a heavy chain having a CDR1 as shown in SEQ ID NO: 155, a CDR2 as shown in SEQ ID NO: 174, a CDR3 as shown in SEQ ID NO: 175, and a light chain having a CDR1 as shown in SEQ ID NO: 114, a CDR2 as shown in SEQ ID NO: 115, a CDR3 as shown in SEQ ID NO: 116; (bl 7) a heavy chain having a FR1 as shown in SEQ ID NO: 258, a CDR1 as shown in SEQ ID NO: 155, an FR2 as shown in SEQ ID NO: 259, a CDR2 as shown in SEQ ID NO: 174, an FR3 as shown in SEQ ID NO: 260, a CDR3 as shown in SEQ ID NO: 175 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 219, a CDR1 as shown in SEQ ID NO: 114, an FR2 as shown in SEQ ID NO: 220, a CDR2 as shown in SEQ ID NO: 115, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 116 and an FR4 as shown in SEQ ID NO: 221;

(cl7) a heavy chain as shown in SEQ ID NO: 173 and a light chain shown in SEQ ID NO: 113; or

(dl7) a heavy chain as defined in (al7) or (bl7), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 173, and a light chain as defined in (al7) or (bl7) and wherein said light chain is at least 80% identical to SEQ ID NO: 113; or

(al 8) a heavy chain having a CDR1 as shown in SEQ ID NO: 155, a CDR2 as shown in SEQ ID NO: 177, a CDR3 as shown in SEQ ID NO: 178, and a light chain having a CDR1 as shown in SEQ ID NO: 118, a CDR2 as shown in SEQ ID NO: 115, a CDR3 as shown in SEQ ID NO: 108;

(bl 8) a heavy chain having a FR1 as shown in SEQ ID NO: 261, a CDR1 as shown in SEQ ID NO: 155, an FR2 as shown in SEQ ID NO: 262, a CDR2 as shown in SEQ ID NO: 177, an FR3 as shown in SEQ ID NO: 263, a CDR3 as shown in SEQ ID NO: 178 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 222, a CDR1 as shown in SEQ ID NO: 118, an FR2 as shown in SEQ ID NO: 223, a CDR2 as shown in SEQ ID NO: 115, an FR3 as shown in SEQ ID NO: 224, a CDR3 as shown in SEQ ID NO: 108 and an FR4 as shown in SEQ ID NO: 61;

(cl8) a heavy chain as shown in SEQ ID NO: 176 and a light chain shown in SEQ ID NO: 117; or

(dl 8) a heavy chain as defined in (al 8) or (bl 8), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 176, and a light chain as defined in (al8) or (b 18) and wherein said light chain is at least 80% identical to SEQ ID NO: 117; or

(al9) a heavy chain having a CDR1 as shown in SEQ ID NO: 180, a CDR2 as shown in SEQ ID NO: 181, a CDR3 as shown in SEQ ID NO: 182, and a light chain having a CDR1 as shown in SEQ ID NO: 120, a CDR2 as shown in SEQ ID NO: 121, a CDR3 as shown in SEQ ID NO: 122;

(bl 9) a heavy chain having a FR1 as shown in SEQ ID NO: 264, a CDR1 as shown in SEQ ID NO: 180, an FR2 as shown in SEQ ID NO: 265, a CDR2 as shown in SEQ ID NO: 181, an FR3 as shown in SEQ ID NO: 266, a CDR3 as shown in SEQ ID NO: 182 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 225, a CDR1 as shown in SEQ ID NO: 120, an FR2 as shown in SEQ ID NO: 223, a CDR2 as shown in SEQ ID NO: 121, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 122 and an FR4 as shown in SEQ ID NO: 61;

(cl9) a heavy chain as shown in SEQ ID NO: 179 and a light chain shown in SEQ ID NO: 119; or

(dl9) a heavy chain as defined in (al9) or (bl9), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 179, and a light chain as defined in (al9) or (bl9) and wherein said light chain is at least 80% identical to SEQ ID NO: 119; or

(a20) a heavy chain having a CDR1 as shown in SEQ ID NO: 149, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 184, and a light chain having a CDR1 as shown in SEQ ID NO: 124, a CDR2 as shown in SEQ ID NO: 125, a CDR3 as shown in SEQ ID NO: 126;

(b20) a heavy chain having a FR1 as shown in SEQ ID NO: 242, a CDR1 as shown in SEQ ID NO: 149, an FR2 as shown in SEQ ID NO: 243, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 182 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 226, a CDR1 as shown in SEQ ID NO: 124, an FR2 as shown in SEQ ID NO: 227, a CDR2 as shown in SEQ ID NO: 125, an FR3 as shown in SEQ ID NO: 228, a CDR3 as shown in SEQ ID NO: 126 and an FR4 as shown in SEQ ID NO: 229;

(c20) a heavy chain as shown in SEQ ID NO: 183 and a light chain shown in SEQ ID NO: 123; or

(d20) a heavy chain as defined in (a20) or (b20), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 183 and a light chain as defined in (a20) or (b20) and wherein said light chain is at least 80% identical to SEQ ID NO: 123; or

(a21) a heavy chain having a CDR1 as shown in SEQ ID NO: 186, a CDR2 as shown in SEQ ID NO: 187, a CDR3 as shown in SEQ ID NO: 190, and a light chain having a CDR1 as shown in SEQ ID NO: 128, a CDR2 as shown in SEQ ID NO: 129, a CDR3 as shown in SEQ ID NO: 130;

(b21) a heavy chain having a FR1 as shown in SEQ ID NO: 77, a CDR1 as shown in SEQ ID NO: 186, an FR2 as shown in SEQ ID NO: 267, a CDR2 as shown in SEQ ID NO: 187, an FR3 as shown in SEQ ID NO: 268, a CDR3 as shown in SEQ ID NO: 190 and an FR4 as shown in SEQ ID NO:72, and a light chain having an FR1 as shown in SEQ ID NO: 230, a CDR1 as shown in SEQ ID NO: 128, an FR2 as shown in SEQ ID NO: 214, a CDR2 as shown in SEQ ID NO: 129, an FR3 as shown in SEQ ID NO: 231, a CDR3 as shown in SEQ ID NO: 130 and an FR4 as shown in SEQ ID NO: 61; (c21) a heavy chain as shown in SEQ ID NO: 185 and a light chain shown in SEQ ID NO: 127; or

(d21) a heavy chain as defined in (a21) or (b21), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 185, and a light chain as defined in (a21) or (b21) and wherein said light chain is at least 80% identical to SEQ ID NO: 127; or

(a22) a heavy chain having a CDR1 as shown in SEQ ID NO: 186, a CDR2 as shown in SEQ ID NO: 187, a CDR3 as shown in SEQ ID NO: 190, and a light chain having a CDR1 as shown in SEQ ID NO: 128, a CDR2 as shown in SEQ ID NO: 129, a CDR3 as shown in SEQ ID NO: 130;

(b22) a heavy chain having a FR1 as shown in SEQ ID NO: 77, a CDR1 as shown in SEQ ID NO: 186, an FR2 as shown in SEQ ID NO: 267, a CDR2 as shown in SEQ ID NO:, 187 an FR3 as shown in SEQ ID NO: 268, a CDR3 as shown in SEQ ID NO: 190 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 230, a CDR1 as shown in SEQ ID NO: 128, an FR2 as shown in SEQ ID NO: 214, a CDR2 as shown in SEQ ID NO: 129, an FR3 as shown in SEQ ID NO: 231, a CDR3 as shown in SEQ ID NO: 130 and an FR4 as shown in SEQ ID NO: 61;

(c22) a heavy chain as shown in SEQ ID NO: 189 and a light chain shown in SEQ ID NO: 127; or

(d22) a heavy chain as defined in (a22) or (b22), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 189, and a light chain as defined in (a22) or (b22) and wherein said light chain is at least 80% identical to SEQ ID NO: 127; or

(a23) a heavy chain having a CDR1 as shown in SEQ ID NO: 192, a CDR2 as shown in SEQ ID NO: 193, a CDR3 as shown in SEQ ID NO: 194, and a light chain having a CDR1 as shown in SEQ ID NO: 132, a CDR2 as shown in SEQ ID NO: 133, a CDR3 as shown in SEQ ID NO: 134;

(b23) a heavy chain having a FR1 as shown in SEQ ID NO: 269, a CDR1 as shown in SEQ ID NO: 192, an FR2 as shown in SEQ ID NO: 270, a CDR2 as shown in SEQ ID NO: 193, an FR3 as shown in SEQ ID NO: 271, a CDR3 as shown in SEQ ID NO: 194 and an FR4 as shown in SEQ ID NO: 76, and a light chain having an FR1 as shown in SEQ ID NO: 232, a CDR1 as shown in SEQ ID NO: 132, an FR2 as shown in SEQ ID NO: 233, a CDR2 as shown in SEQ ID NO: 133, an FR3 as shown in SEQ ID NO: 234, a CDR3 as shown in SEQ ID NO: 134 and an FR4 as shown in SEQ ID NO: 229;

(c23) a heavy chain as shown in SEQ ID NO: 191 and a light chain shown in SEQ ID NO: 131; or (d23) a heavy chain as defined in (a23) or (b23), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 191, and a light chain as defined in (a23) or (b23) and wherein said light chain is at least 80% identical to SEQ ID NO: 131; or

(a24) a heavy chain having a CDR1 as shown in SEQ ID NO: 192, a CDR2 as shown in SEQ ID NO: 193, a CDR3 as shown in SEQ ID NO: 196, and a light chain having a CDR1 as shown in SEQ ID NO: 132, a CDR2 as shown in SEQ ID NO: 133, a CDR3 as shown in SEQ ID NO: 134;

(b24) a heavy chain having a FR1 as shown in SEQ ID NO: 269, a CDR1 as shown in SEQ ID NO: 192, an FR2 as shown in SEQ ID NO: 270, a CDR2 as shown in SEQ ID NO: 193, an FR3 as shown in SEQ ID NO: 271, a CDR3 as shown in SEQ ID NO: 196 and an FR4 as shown in SEQ ID NO: 76, and a light chain having an FR1 as shown in SEQ ID NO: 232, a CDR1 as shown in SEQ ID NO: 132, an FR2 as shown in SEQ ID NO: 233, a CDR2 as shown in SEQ ID NO: 133, an FR3 as shown in SEQ ID NO: 234, a CDR3 as shown in SEQ ID NO: 134 and an FR4 as shown in SEQ ID NO: 229;

(c24) a heavy chain as shown in SEQ ID NO: 195 and a light chain shown in SEQ ID NO: 131; or

(d24) a heavy chain as defined in (a24) or (b24), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 195, and a light chain as defined in (a24) or (b24) and wherein said light chain is at least 80% identical to SEQ ID NO: 131; or

(a25) a heavy chain having a CDR1 as shown in SEQ ID NO: 198, a CDR2 as shown in SEQ ID NO: 199, a CDR3 as shown in SEQ ID NO: 200, and a light chain having a CDR1 as shown in SEQ ID NO: 136, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 137;

(b25) a heavy chain having a FR1 as shown in SEQ ID NO: 272, a CDR1 as shown in SEQ ID NO: 198, an FR2 as shown in SEQ ID NO: 273, a CDR2 as shown in SEQ ID NO: 199, an FR3 as shown in SEQ ID NO: 274, a CDR3 as shown in SEQ ID NO: 200 and an FR4 as shown in SEQ ID NO: 275, and a light chain having an FR1 as shown in SEQ ID NO: 216, a CDR1 as shown in SEQ ID NO: 136, an FR2 as shown in SEQ ID NO: 217, a CDR2 as shown in SEQ ID NO: 111, an FR3 as shown in SEQ ID NO: 235, a CDR3 as shown in SEQ ID NO: 137 and an FR4 as shown in SEQ ID NO: 61;

(c25) a heavy chain as shown in SEQ ID NO: 197 and a light chain shown in SEQ ID NO: 135; or

(d25) a heavy chain as defined in (a25) or (b25), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 197, and a light chain as defined in (a25) or (b25) and wherein said light chain is at least 80% identical to SEQ ID NO: 135; or (a26) a heavy chain having a CDR1 as shown in SEQ ID NO: 202, a CDR2 as shown in SEQ ID NO: 203, a CDR3 as shown in SEQ ID NO: 204, and a light chain having a CDR1 as shown in SEQ ID NO: 139, a CDR2 as shown in SEQ ID NO: 140, a CDR3 as shown in SEQ ID NO: 141;

(b26) a heavy chain having a FR1 as shown in SEQ ID NO: 276, a CDR1 as shown in SEQ ID NO: 202, an FR2 as shown in SEQ ID NO: 277, a CDR2 as shown in SEQ ID NO: 203, an FR3 as shown in SEQ ID NO: 278, a CDR3 as shown in SEQ ID NO: 204 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 236, a CDR1 as shown in SEQ ID NO: 139, an FR2 as shown in SEQ ID NO: 237, a CDR2 as shown in SEQ ID NO: 140, an FR3 as shown in SEQ ID NO: 238, a CDR3 as shown in SEQ ID NO: 141 and an FR4 as shown in SEQ ID NO: 239;

(c26) a heavy chain as shown in SEQ ID NO: 201 and a light chain shown in SEQ ID NO: 138; or

(d26) a heavy chain as defined in (a26) or (b26), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 201, and a light chain as defined in (a26) or (b26) and wherein said light chain is at least 80% identical to SEQ ID NO: 138; or

(a27) a heavy chain having a CDR1 as shown in SEQ ID NO: 202, a CDR2 as shown in SEQ ID NO: 203, a CDR3 as shown in SEQ ID NO: 204, and a light chain having a CDR1 as shown in SEQ ID NO: 139, a CDR2 as shown in SEQ ID NO: 140, a CDR3 as shown in SEQ ID NO: 143;

(b27) a heavy chain having a FR1 as shown in SEQ ID NO: 276, a CDR1 as shown in SEQ ID NO: 202, an FR2 as shown in SEQ ID NO: 277, a CDR2 as shown in SEQ ID NO: 203, an FR3 as shown in SEQ ID NO: 278, a CDR3 as shown in SEQ ID NO: 204 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 236, a CDR1 as shown in SEQ ID NO: 139, an FR2 as shown in SEQ ID NO: 237, a CDR2 as shown in SEQ ID NO: 140, an FR3 as shown in SEQ ID NO: 238, a CDR3 as shown in SEQ ID NO: 143 and an FR4 as shown in SEQ ID NO: 61;

(c27) a heavy chain as shown in SEQ ID NO: 201 and a light chain shown in SEQ ID NO: 141; or

(d27) a heavy chain as defined in (a27) or (b27), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 201, and a light chain as defined in (a27) or (b27) and wherein said light chain is at least 80% identical to SEQ ID NO: 141; or

(a28) a heavy chain having a CDR1 as shown in SEQ ID NO: 206, a CDR2 as shown in SEQ ID NO: 207, a CDR3 as shown in SEQ ID NO: 208, and a light chain having a CDR1 as shown in SEQ ID NO: 145, a CDR2 as shown in SEQ ID NO: 146, a CDR3 as shown in SEQ ID NO: (b28) a heavy chain having a FR1 as shown in SEQ ID NO: 279, a CDR1 as shown in SEQ ID NO: 206, an FR2 as shown in SEQ ID NO: 280, a CDR2 as shown in SEQ ID NO: 207, an FR3 as shown in SEQ ID NO: 281, a CDR3 as shown in SEQ ID NO: 208 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 285, a CDR1 as shown in SEQ ID NO: 145, an FR2 as shown in SEQ ID NO: 240, a CDR2 as shown in SEQ ID NO: 146, an FR3 as shown in SEQ ID NO: 241, a CDR3 as shown in SEQ ID NO: 147 and an FR4 as shown in SEQ ID NO: 61;

(c28) a heavy chain as shown in SEQ ID NO: 205 and a light chain shown in SEQ ID NO: 144; or

(d28) a heavy chain as defined in (a) or (b), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 205, and a light chain as defined in (a) or (b) and wherein said light chain is at least 80% identical to SEQ ID NO: 144.

Particularly preferred is an antibody as defined in (alO) to (dlO), (al4) to (dl4), (a20) to (d20), (a21) to (d21(, (a23) to (d23), (a24) to (d24), (a26) to (d26) and (a27) to (d27).

The present invention further relates to a VAST-bound immunogen as described herein elsewhere for use as a vaccine, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP) wherein sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Preferably, the vaccine is suitable for preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma.

The term “vaccine” as used herein, refers to a composition comprising an immunogen which, when inoculated into a subject, has the effect of stimulating a cellular immune response comprising a T-cell response and/or a humoral immune response comprising a B-cell response generally resulting in antibody production. The T cell response can be a cytotoxic T-cell response directed against a cell that expresses the immunogen. However, the induction of a T- cell response comprising other types of T cells by an immunogen disclosed herein is also contemplated. A B-cell response results in the production of antibody that binds to the immunogen. The vaccine can serve to elicit an immune response in the mammal which serves to protect the mammal against a disease. The term "vaccine" does not in any way connote that the composition is capable of fully preventing a disease in a vaccinated subject, or providing any specific or general level of protection against a disease. A vaccine can be ineffective in certain subjects while inducing an immune response in other subjects. Preferably, the vaccine according to the present invention refers in this context to a VAST-bound immunogen that provides active acquired immunity to a malignant disease, e.g. cancer associated with aberrant MUC1 expression.

The vaccine may include one or more adjuvants. As used herein the term “adjuvant” refers to a compound that, when used in combination with a specific antigenic particle in a formulation, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response can include intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.

Examples of known adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response, containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminium hydroxide adjuvant. Other known adjuvants include granulocyte macrophage colony-stimulating factor (GM-CSF), Bacillus Calmette-Guerin (BCG), aluminium hydroxide, Muramyl dipeptide (MDP) compounds, such as thur-MDP and nor- MDP, muramyl tripeptide phosphatidylethanolamine (MTP-PE), RIBI's adjuvants (Ribi ImmunoChem Research, Inc., Hamilton Mont.), which contains three components extracted from bacteria, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MF-59, Novasomes®, major histocompatibility complex (MHC) antigens are other known adjuvants.

The vaccine of the present invention may also be formulated as described for a pharmaceutical composition elsewhere herein.

The present invention also refers to a method for manufacturing an antibody that specifically binds to hypoglycosylated MUC1, said method comprising the step of obtaining the antibody from a sample of an animal which has been immunized with a VAST-bound immunogen, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

The term “manufacture” as used herein refers to the process of generation of the antibody which specifically binds to hypoglycosylated MUC1 in a host cell. The manufacture may also comprise further steps such as purifying the produced antibody or formulating the antibody or purified antibody as a pharmaceutical composition. Accordingly, the aforementioned method of the present invention may consist of the aforementioned steps or may comprise further additional steps.

Preferably, the animal in the aforementioned method for manufacturing an antibody has been immunized using an immunization scheme having the following steps: a) two times priming using VAST-bound immunogen; b) two times boosting using VAST-bound immunogen and free immunogen; and c) one time boosting using free immunogen.

Preferably, the time between i) the first and second priming in step a) is about 4 weeks, ii) the second priming in step a) and the first boosting in step b) is about 4 weeks to about 6 weeks, iii) the first and the second boosting in step b) is about 4 weeks and iv) the second boosting in step b) and the boosting in step c) is about 4 weeks.

The antibody of the present invention is obtained from a sample of an animal. Preferably, the sample refers to a bodyfluid sample of the animal which has been immunized as described herein elsewhere, preferably, blood, plasma, or serum, or any combination thereof. Other typical samples include any bodyfluid obtained from a mammalian subject, tissue biopsy, sputum, lymphatic fluid, blood cells (e.g., peripheral blood mononuclear cells), tissue or fine needle biopsy samples.

The antibodies shall not only be obtained from a single individual animal but rather may be a mixture of different antibodies taken from different animals. The antibodies obtained from the immunized animal form a polyclonal population of antibodies, since different B cells produce members of said population. Preferably, this polyclonal population specifically binds the immunogen as described herein elsewhere. It should be noted that within the polyclonal population, at least one antibody specifically binds to the immunogen, however, that one antibody is not necessarily isolated from the other antibodies within the polyclonal population that do not specifically bind to the antigen. Preferably, more than one different antibody within the polyclonal population specifically binds to the immunogen of the present invention.

It is also contemplated to obtain the antibodies from a tissue culture supernatant of B cells grown in vitro. For example, a population of B cells may be obtained from an animal that has been subjected to the immunogen as described herein elsewhere. Said population may then be expanded, e.g. to enrich B cells in the population as compared to other white blood cells. From the cultured media of these cells (into which the polyclonal antibodies are secreted), the polyclonal population of antibodies can be isolated.

The antibodies of the present invention may be obtained by purifying or partially purifying the antibodies from proteins, polypeptides or other contaminants that would interfere with the therapeutic, diagnostic or other use of the antibodies. For example, the polyclonal antibodies can be purified using any conventional purification technology including precipitation, filtration, ultra-filtration, extraction, chromatography techniques such as ion-exchange-, affinity- and/or size exclusion chromatography, HPLC, dialysis or electrophoresis. The skilled person is well aware of how an antibody may be purified in order to provide it in isolated form. Antibody molecules may be purified via affinity purification with proteins/ligands that specifically and reversibly bind constant domains such as the CHI or the CL domains. Examples of such proteins are immunoglobulin-binding bacterial proteins such as Protein A, Protein G, Protein A/G or Protein L. It is also possible to equip one of the chains of the antibody molecule of the invention with one or more affinity tags. Affinity tags such as the Strep-tag® or Strep-tag® II, the myc-tag, the FLAG™-tag, the His6-tag or the HA-tag allow easy detection and also simple purification of the antibodies.

The method of manufacturing the antibody of the present invention may also, preferably, encompass immunizing an animal as described herein elsewhere and sacrificing said animal.

The antibodies of the present invention may also be manufactured by immunizing animals using an immunization scheme as described herein elsewhere and sequencing the antibody repertoire generated in response to said immunization scheme. Preferably, single-cell sequencing of B cells shall be used to identify endogenously paired variable heavy and variable light chain regions from single B cells, which can be used to subsequently reconstruct antibodies.

The following are particular preferred embodiments of the present invention.

Embodiment 1 : An antibody that specifically binds to hypoglycosylated MUC1, wherein said antibody binds to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein.

Embodiment 2: The antibody of embodiment 1, wherein said carbohydrate moiety is sialyated N-acetylgalactosamine (GalNAc) linked to a serine or threonine residue in the peptide backbone portion by a glycosidic bond. Embodiment 3: The antibody of embodiment 1 or 2, wherein said peptide portion comprises the amino acid sequence shown in SEQ ID NO: 95 (PGSTA).

Embodiment 4: The antibody of embodiment 3, wherein said sialyated N-acetylgalactosamine (GalNAc) is linked to the serine or threonine residue in SEQ ID NO: 95 by a glycosidic bond.

Embodiment 5: The antibody of any one of embodiments 1 to 4, wherein said hypoglycoslyated MUC1 is a cancer specific MUC1, preferably, an adenocarcinoma specific MUC1.

Embodiment 6: The antibody of any one of embodiments 1 to 5, wherein said antibody is a polyclonal or monoclonal antibody.

Embodiment 7: The antibody of any one of embodiments 1 to 6, wherein the antibody is obtainable by immunizing an animal with a VAST-bound immunogen, wherein said VAST- bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Embodiment 8: The antibody of any one of embodiments 1 to 7, wherein said antibody is obtainable by immunizing an animal using an immunization scheme having the following steps: a) two times priming using VAST-bound immunogen; b) two times boosting using VAST-bound immunogen and free immunogen; and c) one time boosting using free immunogen.

Embodiment 9: The antibody of embodiment 8, wherein the time between i) the first and second priming in step a) is about 4 weeks, ii) the second priming in step a) and the first boosting in step b) is about 4 weeks to about 6 weeks, iii) the first and the second boosting in step b) is about 4 weeks and iv) the second boosting in step b) and the boosting in step c) is about 4 weeks.

Embodiment 10: An antibody as defined in any one of embodiments 1 to 9 for use in treating and/or preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma.

Embodiment 11 : An antibody, preferably according to any one of embodiments 1 to 10, comprising: (al) a heavy chain having a CDR1 as shown in SEQ ID NO: 24, a CDR2 as shown in SEQ ID NO: 25, a CDR3 as shown in SEQ ID NO 26, and a light chain having a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO 4;

(cl) a heavy chain as shown in SEQ ID NO: 23 and a light chain shown in SEQ ID NO: 1; or (dl) a heavy chain as defined in (al) or (bl) and wherein said heavy chain is at least 80% identical to SEQ ID NO: 23, and a light chain as defined in (al) or (bl) and wherein said light chain is at least 80% identical to SEQ ID NO: 1; or

(c2) a heavy chain as shown in SEQ ID NO: 27 and a light chain shown in SEQ ID NO: 5; or (d2) a heavy chain as defined in (al) or (bl), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 27, and a light chain as defined in (a2) or (b2) and wherein said light chain is at least 80% identical to SEQ ID NO: 5; or

(a4) a heavy chain having a CDR1 as shown in SEQ ID NO: 36, a CDR2 as shown in SEQ ID NO: 29, a CDR3 as shown in SEQ ID NO: 37, and a light chain having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10, a CDR3 as shown in SEQ ID NO 11;

(b4) a heavy chain having a FR1 as shown in SEQ ID NO: 80, a CDR1 as shown in SEQ ID NO: 36, an FR2 as shown in SEQ ID NO: 74, a CDR2 as shown in SEQ ID NO: 29, an FR3 as shown in SEQ ID NO: 81, a CDR3 as shown in SEQ ID NO: 37 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 54, a CDR1 as shown in SEQ ID NO: 9, an FR2 as shown in SEQ ID NO: 55, a CDR2 as shown in SEQ ID NO: 10, an FR3 as shown in SEQ ID NO: 56, a CDR3 as shown in SEQ ID NO: 11 and an FR4 as shown in SEQ ID NO: 57;

(c4) a heavy chain as shown in SEQ ID NO: 35 and a light chain shown in SEQ ID NO: 8; or (d4) a heavy chain as defined in (a4) or (b4), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 35, and a light chain as defined in (a4) or (b4) and wherein said light chain is at least 80% identical to SEQ ID NO: 8; or

(b5) a heavy chain having a FR1 as shown in SEQ ID NO: 83, a CDR1 as shown in SEQ ID NO: 39, an FR2 as shown in SEQ ID NO: 84, a CDR2 as shown in SEQ ID NO: 40, an FR3 as shown in SEQ ID NO: 85, a CDR3 as shown in SEQ ID NO: 41 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 58, a CDR1 as shown in SEQ ID NO:, an FR2 as shown in SEQ ID NO: 59, a CDR2 as shown in SEQ ID NO:, an FR3 as shown in SEQ ID NO: 60, a CDR3 as shown in SEQ ID NO: and an FR4 as shown in SEQ ID NO: 61; (c5) a heavy chain as shown in SEQ ID NO: 38 and a light chain shown in SEQ ID NO: 12; or

(d5) a heavy chain as defined in (a5) or (b5), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 38, and a light chain as defined in (a5) or (b5) and wherein said light chain is at least 80% identical to SEQ ID NO: 12; or

(a6) a heavy chain having a CDR1 as shown in SEQ ID NO: 43, a CDR2 as shown in SEQ ID NO: 44, a CDR3 as shown in SEQ ID NO: 45, and a light chain having a CDR1 as shown in SEQ ID NO: 16, a CDR2 as shown in SEQ ID NO: 17, a CDR3 as shown in SEQ ID NO: 18;

(c7) a heavy chain as shown in SEQ ID NO: 46 and a light chain shown in SEQ ID NO: 19; or (d7) a heavy chain as defined in (a7) or (b7), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 46, and a light chain as defined in (a7) or (b7) and wherein said light chain is at least 80% identical to SEQ ID NO: 19; or

(a8) a heavy chain having a CDR1 as shown in SEQ ID NO: 149, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 151, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 102, a CDR3 as shown in SEQ ID NO: 103;

(b8) a heavy chain having a FR1 as shown in SEQ ID NO: 242, a CDR1 as shown in SEQ ID NO: 149, an FR2 as shown in SEQ ID NO: 243, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 151 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 209, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 102, an FR3 as shown in SEQ ID NO: 211 a CDR3 as shown in SEQ ID NO: 103 and an FR4 as shown in SEQ ID NO: 212;

(d9) a heavy chain as defined in (a9) or (b9), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 152, and a light chain as defined in (a9) or (b9) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or (alO) a heavy chain having a CDR1 as shown in SEQ ID NO: 155, a CDR2 as shown in SEQ ID NO: 33, a CDR3 as shown in SEQ ID NO: 156, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 102, a CDR3 as shown in SEQ ID NO: 103;

(dl 1) a heavy chain as defined in (al l) or (bl 1), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 157, and a light chain as defined in (al 1) or (bl 1) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(cl 2) a heavy chain as shown in SEQ ID NO: 161 and a light chain shown in SEQ ID NO: 104; or

(dl2) a heavy chain as defined in (al2) or (bl2), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 161, and a light chain as defined in (al2) or (bl2) and wherein said light chain is at least 80% identical to SEQ ID NO: 104; or

(al 3) a heavy chain having a CDR1 as shown in SEQ ID NO: 158 , a CDR2 as shown in SEQ ID NO: 159, a CDR3 as shown in SEQ ID NO: 160, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO: 4;

(bl 3) a heavy chain having a FR1 as shown in SEQ ID NO: 249, a CDR1 as shown in SEQ ID NO: 158, an FR2 as shown in SEQ ID NO: 250, a CDR2 as shown in SEQ ID NO: 159, an FR3 as shown in SEQ ID NO: 251, a CDR3 as shown in SEQ ID NO: 160 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(cl 4) a heavy chain as shown in SEQ ID NO: 167 and a light chain shown in SEQ ID NO: 105; or

(dl4) a heavy chain as defined in (al4) or (bl4), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 167, and a light chain as defined in (al4) or (bl4) and wherein said light chain is at least 80% identical to SEQ ID NO: 105; or

(al5) a heavy chain having a CDR1 as shown in SEQ ID NO: 283, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 284, and a light chain having a CDR1 as shown in SEQ ID NO: 110, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 112;

(cl5) a heavy chain as shown in SEQ ID NO: 282 and a light chain shown in SEQ ID NO: 109; or

(dl 5) a heavy chain as defined in (al 5) or (bl 5), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 282, and a light chain as defined in (al 5) or (bl 5) and wherein said light chain is at least 80% identical to SEQ ID NO: 109; or

(bl6) a heavy chain having a FR1 as shown in SEQ ID NO: 256, a CDR1 as shown in SEQ ID NO: 283, an FR2 as shown in SEQ ID NO: 257, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 172 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 216, a CDR1 as shown in SEQ ID NO: 110, an FR2 as shown in SEQ ID NO: 217, a CDR2 as shown in SEQ ID NO: 111, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 112 and an FR4 as shown in SEQ ID NO: 61; (cl6) a heavy chain as shown in SEQ ID NO: 171 and a light chain shown in SEQ ID NO: 109; or

(al7) a heavy chain having a CDR1 as shown in SEQ ID NO: 155, a CDR2 as shown in SEQ ID NO: 174, a CDR3 as shown in SEQ ID NO: 175, and a light chain having a CDR1 as shown in SEQ ID NO: 114, a CDR2 as shown in SEQ ID NO: 115, a CDR3 as shown in SEQ ID NO: 116;

(bl 7) a heavy chain having a FR1 as shown in SEQ ID NO: 258, a CDR1 as shown in SEQ ID NO: 155, an FR2 as shown in SEQ ID NO: 259, a CDR2 as shown in SEQ ID NO: 174, an FR3 as shown in SEQ ID NO: 260, a CDR3 as shown in SEQ ID NO: 175 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 219, a CDR1 as shown in SEQ ID NO: 114, an FR2 as shown in SEQ ID NO: 220, a CDR2 as shown in SEQ ID NO: 115, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 116 and an FR4 as shown in SEQ ID NO: 221;

(cl 7) a heavy chain as shown in SEQ ID NO: 173 and a light chain shown in SEQ ID NO: 113; or

(cl8) a heavy chain as shown in SEQ ID NO: 176 and a light chain shown in SEQ ID NO: 117; or (dl 8) a heavy chain as defined in (al 8) or (bl 8), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 176, and a light chain as defined in (al8) or (b 18) and wherein said light chain is at least 80% identical to SEQ ID NO: 117; or

(bl 9) a heavy chain having a FR1 as shown in SEQ ID NO: 264, a CDR1 as shown in SEQ ID NO: 180, an FR2 as shown in SEQ ID NO: 265, a CDR2 as shown in SEQ ID NO: 181, an FR3 as shown in SEQ ID NO: 266, a CDR3 as shown in SEQ ID NO: 182 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO:

225, a CDR1 as shown in SEQ ID NO: 120, an FR2 as shown in SEQ ID NO: 223, a CDR2 as shown in SEQ ID NO: 121, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 122 and an FR4 as shown in SEQ ID NO: 61;

(b20) a heavy chain having a FR1 as shown in SEQ ID NO: 242, a CDR1 as shown in SEQ ID NO: 149, an FR2 as shown in SEQ ID NO: 243, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 182 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO:

226, a CDR1 as shown in SEQ ID NO: 124, an FR2 as shown in SEQ ID NO: 227, a CDR2 as shown in SEQ ID NO: 125, an FR3 as shown in SEQ ID NO: 228, a CDR3 as shown in SEQ ID NO: 126 and an FR4 as shown in SEQ ID NO: 229;

(d20) a heavy chain as defined in (a20) or (b20), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 183 and a light chain as defined in (a20) or (b20) and wherein said light chain is at least 80% identical to SEQ ID NO: 123; or (a21) a heavy chain having a CDR1 as shown in SEQ ID NO: 186, a CDR2 as shown in SEQ ID NO: 187, a CDR3 as shown in SEQ ID NO: 190, and a light chain having a CDR1 as shown in SEQ ID NO: 128, a CDR2 as shown in SEQ ID NO: 129, a CDR3 as shown in SEQ ID NO: 130;

(b21) a heavy chain having a FR1 as shown in SEQ ID NO: 77, a CDR1 as shown in SEQ ID NO: 186, an FR2 as shown in SEQ ID NO: 267, a CDR2 as shown in SEQ ID NO: 187, an FR3 as shown in SEQ ID NO: 268, a CDR3 as shown in SEQ ID NO: 190 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 230, a CDR1 as shown in SEQ ID NO: 128, an FR2 as shown in SEQ ID NO: 214, a CDR2 as shown in SEQ ID NO: 129, an FR3 as shown in SEQ ID NO: 231, a CDR3 as shown in SEQ ID NO: 130 and an FR4 as shown in SEQ ID NO: 61;

(c21) a heavy chain as shown in SEQ ID NO: 185 and a light chain shown in SEQ ID NO: 127; or

(a23) a heavy chain having a CDR1 as shown in SEQ ID NO: 192, a CDR2 as shown in SEQ ID NO: 193, a CDR3 as shown in SEQ ID NO: 194, and a light chain having a CDR1 as shown in SEQ ID NO: 132, a CDR2 as shown in SEQ ID NO: 133, a CDR3 as shown in SEQ ID NO: 134; (b23) a heavy chain having a FR1 as shown in SEQ ID NO: 269, a CDR1 as shown in SEQ ID NO: 192, an FR2 as shown in SEQ ID NO: 270, a CDR2 as shown in SEQ ID NO: 193, an FR3 as shown in SEQ ID NO: 271, a CDR3 as shown in SEQ ID NO: 194 and an FR4 as shown in SEQ ID NO: 76, and a light chain having an FR1 as shown in SEQ ID NO: 232, a CDR1 as shown in SEQ ID NO: 132, an FR2 as shown in SEQ ID NO: 233, a CDR2 as shown in SEQ ID NO: 133, an FR3 as shown in SEQ ID NO: 234, a CDR3 as shown in SEQ ID NO: 134 and an FR4 as shown in SEQ ID NO: 229;

(c23) a heavy chain as shown in SEQ ID NO: 191 and a light chain shown in SEQ ID NO: 131; or

(d23) a heavy chain as defined in (a23) or (b23), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 191, and a light chain as defined in (a23) or (b23) and wherein said light chain is at least 80% identical to SEQ ID NO: 131; or

(d25) a heavy chain as defined in (a25) or (b25), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 197, and a light chain as defined in (a25) or (b25) and wherein said light chain is at least 80% identical to SEQ ID NO: 135; or

(a26) a heavy chain having a CDR1 as shown in SEQ ID NO: 202, a CDR2 as shown in SEQ ID NO: 203, a CDR3 as shown in SEQ ID NO: 204, and a light chain having a CDR1 as shown in SEQ ID NO: 139, a CDR2 as shown in SEQ ID NO: 140, a CDR3 as shown in SEQ ID NO: 141;

(b27) a heavy chain having a FR1 as shown in SEQ ID NO: 276, a CDR1 as shown in SEQ ID NO: 202, an FR2 as shown in SEQ ID NO: 277, a CDR2 as shown in SEQ ID NO: 203, an FR3 as shown in SEQ ID NO: 278, a CDR3 as shown in SEQ ID NO: 204 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 236, a CDR1 as shown in SEQ ID NO: 139, an FR2 as shown in SEQ ID NO: 237, a CDR2 as shown in SEQ ID NO: 140, an FR3 as shown in SEQ ID NO: 238, a CDR3 as shown in SEQ ID NO: 143 and an FR4 as shown in SEQ ID NO: 61; (c27) a heavy chain as shown in SEQ ID NO: 201 and a light chain shown in SEQ ID NO: 141; or

(a28) a heavy chain having a CDR1 as shown in SEQ ID NO: 206, a CDR2 as shown in SEQ ID NO: 207, a CDR3 as shown in SEQ ID NO: 208, and a light chain having a CDR1 as shown in SEQ ID NO: 145, a CDR2 as shown in SEQ ID NO: 146, a CDR3 as shown in SEQ ID NO: 147;

(b28) a heavy chain having a FR1 as shown in SEQ ID NO: 279, a CDR1 as shown in SEQ ID NO: 206, an FR2 as shown in SEQ ID NO: 280, a CDR2 as shown in SEQ ID NO: 207, an FR3 as shown in SEQ ID NO: 281, a CDR3 as shown in SEQ ID NO: 208 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 285, a CDR1 as shown in SEQ ID NO: 145, an FR2 as shown in SEQ ID NO: 240, a CDR2 as shown in SEQ ID NO: 146, an FR3 as shown in SEQ ID NO: 241, a CDR3 as shown in SEQ ID NO: 147 and an FR4 as shown in SEQ ID NO: 61;

Embodiment 12: A VAST-bound immunogenfor use as a vaccine, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP) wherein sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Embodiment 13: The VAST-bound immunogen for use of embodiment 12, wherein said vaccine is suitable for preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma.

Embodiment 14: A method for manufacturing an antibody that specifically binds to hypoglycosylated MUC1, said method comprising the step of obtaining the antibody from a sample of an animal which has been immunized with a VAST-bound immunogen, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N- acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

Embodiment 15: The method of embodiment 14, wherein said animal has been immunized using an immunization scheme having the following steps: a) two times priming using VAST-bound immunogen; b) two times boosting using VAST-bound immunogen and free immunogen; and c) one time boosting using free immunogen.

Embodiment 16: The method of embodiment 15, wherein the time between i) the first and second priming in step a) is about 4 weeks, ii) the second priming in step a) and the first boosting in step b) is about 4 weeks to about 6 weeks, iii) the first and the second boosting in step b) is about 4 weeks and iv) the second boosting in step b) and the boosting in step c) is about 4 weeks.

All references cited throughout this specification are herewith incorporated by reference in their entirety as well as with respect to the specifically mentioned disclosure content.

FIGURES

Figure 1: Visual representation of the difference between normal MUC-1 present on healthy cells and the tumor specific MUC-1 present on cancer cells. (A) Healthy cells display “normal MUC-1.” In this context, the long and flexible extracellular domain of the protein is heavily glycosylated at both N and O-linked positions. However, in many solid tumor cancers, the MUC-1 displayed by epithelial cells is heterogeneously hypoglycosylated. The truncated glycans are often pre-maturely siaylated, leading to the presentation of core and/or abnormal glycan epitopes. These epitopes can be targeted by several different categories of antibody. When immunizing animals with a heterogeneous mixture of MUC-1 variants, all categories of antibody are elicited with no focus applied to any given antibody species. Our analysis of the literature suggests that antibodies from category 3 (those that are true glycopeptide binders) are the most favorable. For this reason, we have designed immunogens and screening assays that should facilitate the production of specifically that type of antibody. (B) Alternative visualization of MUC-1, which illustrates the domain architecture of the protein as well as a non-exhaustive list of VNTR-binding monoclonal antibodies that have been generated by others in the past. Many of these antibodies have been used in clinical trials, although so far none have succeeded. Figure 2: VAST generation and immunization. (A) MUC-1 peptides were sortagged onto the VAST. VAST preparation was validated by fluorescent lectin staining (since the MUC-1 peptides carry specific glycans) and visualized by flow cytometry. Helix pomatia lectin stains siaylated Tn equally well irrespective of which amino acid the glycan is attached to (left). Sambucus nigra lectin recognizes STn but has a clear preference for the glycan when bound to a Threonine residue. The consistent results illustrate that MUC-1 VASTs were successfully generated. (B) MUC-1 VAST immunization was performed in mice according to the depicted injection schedule. Mice were initially primed (P) with two injections of membranous VAST. The highly-avid membranous VAST is designed to facilitate the establishment of memory B cells from even low affinity and/or rare naive B cell precursors. Mice were then boosted (B) three times using first a combination of membranous VAST and soluble VSG protein, followed by a final boost of soluble VSG protein. This boosting strategy simultaneously facilitates the continued establishment of additional memory populations via the membranous VAST, and the expansion/differentiation/affmity maturation of the existing memory populations via the soluble di- or tri-valent VSG protein (10-million fold less avid).

Figure 3: Mice immunized with MUC-1 VASTs develop high titers to MUC-1 immunogens. Top: Shown are the sequence of the MUC-1 immunogen peptide and the shorter peptide used to assess antibody titer level. Using this strategy, we immunize with the full VNTR region of MUC-1, while only assessing whether the particular region of interest (surrounding the glycan) is being targeted. Bottom: The graph depicts the results of an ELISA assay; serum from 5 immunized mice per group (mouse/M 1-5) were titered against the cognate peptides (i.e., mice immunized with MUC-1 STn-Serine VASTs were titered against the MUC-1 STn- Serine peptide). The “baseline bleed” is the titer level observed when pre-immune serum was used in the assay. This titer level is the background. In grey are the “final bleed” samples, taken after the final immunization. The dramatic increase in ELISA signal from baseline to final bleed in 8/10 mice reveals that the majority of the immunized animals developed strong antibody responses.

Figure 4: Mice immunized with MUC-1 STn-Serine VASTs develop polyclonal titers that are exsquisitely specific to the STn-Serine epitope. In this ELISA assay, the “final bleed” serum from mice immunized with MUC-1 STn-Serine VASTs (shown in Fig. 4; left) were titered against the cognate peptide as well as two extremely similar peptides. The ELISA bait peptides are conceptually described in the image on the right side of the graph, as well as in the ELISA legend. Consistent with the results in Fig. 4 (left), 3 out of 5 immunized mice displayed cognate peptide-reactive antibodies. However, none of the immunized mice displayed titers that bound to the similar peptides. Figure 5. Mice immunized with MUC-1 STn-Threonine VASTs develop polyclonal titers that are exquisitely specific a variety of MUC-1 epitopes. (A) In this ELISA assay, the “final bleed” serum from mice (mouse/M 1-5) immunized with MUC-1 STn-Threonine VASTs (shown in Fig. 3; right) were titered against the cognate peptide as well as two extremely similar peptides. The ELISA bait peptides are conceptually described in the image on the right side of the graph, as well as in the ELISA legend. Consistent with the results in Fig. 3 (right), 5 out of 5 immunized mice displayed cognate peptide-reactive antibodies (lightest grey). Ml displayed titers that were capable of binding with equal efficiency to STn-Threonine and Tn-Threonine- containing MUC-1 glycopeptides. M2, 3, and 4 displayed titers that were capable of binding with similar efficiencies to STn-Threonine and STn-Serine-containing MUC-1 glycopeptides. Finally, M5 displayed titers that were specific to the STn-Threonine-containing MUC-1 glycopeptide. (B) A similar ELISA is shown. In this case, the antisera is again tittered against the cognate glycopeptide (“S-TnThr”) in addition to the same peptide without a glycan, illustrating that the presence of the glycan is critical for antibody binding (with the exception of Ml, similarly as observed in Fig. 5A).

Figure 6. Anti-MUC-1 antisera samples recognize MUC-1 on tumor cells. Shown are flow cytometry plots, wherein antisera samples were incubated with OvCar 3 cells (a tumor cell line well-known to display hypoglycosylated variants of MUC-1) and then detected using a fluorescent anti-mouse IgG antibody. (A) Cells were stained with a control antiserum sample (antisera collected from a mouse immunized with VAST that was not sortagged with a MUC- 1 peptide) and an STn-SER-responding antiserum sample. (B) The same control sample is shown but now compared to an STn-THR-responding antiserum sample. (C) Illustrated here is an independent replicate of the same experiment that now includes several additional control conditions.

Figure 7. Identification and isolation of B cells expressing antigen-binding B cell receptors. (A) A simplified schematic of the experimental workflow is shown. Splenocytes from immunized mice are isolated and homogenized. A fluorescent version of the antigen of interest is then incubated with the cells (along with a series of well-known B cell identifying antibodies - not shown). The cells are subjected to FACS analysis, whereby antigen-positive B cells are isolated from the total population and ultimately sorted into 384-well plates with a singular cell per well. These isolated cells are then further characterized through downstream approaches. (B) Representative data illustrating the efficiency of cell isolation is shown. Here, splenocytes from a mouse immunized with sTn-SER-MUC-1 VASTs were homogenized and incubated with a shortened version of the immunized peptide (similar to that which is used in figures 5 and 6 except that the Cy5 fluorophore is also incorporated at the N-terminal end). The FACS plot illustrates the efficiency of cell identification. Only the cells that show positive staining results for both the Cy5 label and a streptavidin-based fluorescent label are of interest (gated and labeled as “Cy5⁺bio⁺”). 1.31% of the total B cell population falls into this category.

Figure 8. Candidate antibodies identified. Shown is a circos plot. The plot depicts certain characteristics of a given antibody being expressed by a given B cell. The heavy and light chain gene combinations (V, D, and J genes) expressed by each isolated candidate B cell are shown. The heavy chain combinations are shown on the bottom half of the plot, with the ribbon across the plot connecting to the light chain gene combination present in a single given cell which are shown across the top. Some of the highest quality candidates are marked with asterisks. (*) marks a set of antibody candidates that each share the same light chain. (**) marks a pair of clonal cells, displaying identical gene and CDR3 combinations. These candidate antibodies were identified in STn-Ser immunized mice.

Figure 9. Several of the high quality BCR candidates display excessive somatic hypermutations and high binding capacity. Shown are FACS plots depicting the same type of data displayed in Fig. 6B, except that the represented cells are now only those that are described in Fig. 8. Each circle on the plots denotes an individual baited B cell, while the sizes of the circles (see legend) represent the number of somatic hypermutations detected within that BCR’s heavy chain (A) and light chain (B) relative to the germline sequences of those V, D, and J genes. Cells that have high numbers of somatic hypermutations and/or cells that appear closest to the top right quadrant of the plots are those that express the highest quality candidate BCRs.

Figure 10. STn-Threonine-elicited antisera display strong marks of class switch recombination. The ELISA plot illustrates antisera tittering against the indicated glycopeptides after resolving with either an anti-mouse IgM secondary antibody or an anti-mouse IgG secondary antibody. IgG antibodies are typically expected to be of higher affinity than IgM antibodies after the B cell producing said antibody undergoes germinal center reactions, leading to both class switching and somatic hypermutation. High IgG-specific titers therefore indicate successful immunization and high-affinity antibody elicitation.

Figure 11. Additional candidate antibodies identified. (A) Data illustrated here is conceptually similar to the data shown in Fig. 9, except that these antibodies were isolated from STn-Thr immunized mice. (B) Highlighted are two sets of antibody pairs that display identical heavy and light chain VDJ recombination patters, despite originating in two different mice (mouse/”M” 3 and mouse/”M” 4). Detection of such patterns is further evidence that high quality antibodies are present in the dataset. Figure 12. B cell characterization from STn-Thr immunized mice. (A) Shown is a flow cytometry plot of all sorted and sequenced B cells isolated from the STn-Thr immunized mice according to the strategy described in Fig. 8A. Several of the cells that were selected for mAh characterization have their VDJ recombination pattern overlayed on the plot. The X and Y axes denote the fluorescent signals exhibited by each B cell from the two types of STn-Thr bait reagents. (B) UMAP plot illustrates the B cell subpopulation identifications determined by RNA seq analysis. The memory B cell population is likely to contain the highest proportion of high quality and affinity matured antibodies.

Figure 13. Monoclonal antibody production. (A) Schematic of the overall antibody production workflow deployed here. Antibodies are produced in either HEK or CHO cells after transfection with separate vectors encoding the complete heavy chain and light chain of each antibody. The antibodies are secreted into the supernatant of the expressor cell lines. Antibodies are purified from the media by protein G affinity chromatography before being analyzed by several quality control measures. (B) Example data illustrating the results of the purification strategy. SDS PAGE and Coomassie stained gel shows the reduced heavy and light chains appearing at the expected molecular weights for a panel of antibodies.

Figure 14. mAb 135 binds to MUC-1. ELISA illustrates the binding of monovalent Fab- versions of mAb 135, PankomAb, and an internal non-MUC-1 binding antibody of the same isotype. mAb 135 binds strongly to the STn-Thr glycopeptide, while it does not bind to the sugarless peptide as expected. PankomAb, an antibody that has been used in human clinical trials, does not appear to bind to MUC-1 glycopeptides when produced as a Fab (though it does bind strongly when produced as a bivalent IgG). This suggests that mAb 135 has a notably higher binding affinity.

Figure 15. The isolated antibodies bind to human tumor cells. (A) The flow cytometry plot illustrates the binding of several discovered antibodies to tumor-associated MUC-1 -expressing OvCar-3 cells. Again, several of the antibodies bind more strongly than the commercially available comparator antibody PankomAb. (B) Immunofluorescence stains of human ovarian SKOV-3 cells, which also express tumor associated MUC-1, using some of the discovered antibodies. DAPI stain denotes the location of the nucleus of each cell in the frame, which is used just as a proxy to indicate both cell location and cell density. These data further support the conclusion that the dataset of isolated mAbs are of high-quality.

Figure 16. Anti-MUCl antibodies are endocytosed by tumor cells. (A) The images depict the fluorescence profile of cell populations treated for 18 hours with indicated concentrations of a MUC-1 antibody that was labeled with pHPhrodo green, a fluorescent dye that emits fluorescence in this channel only when acidified. Fluorescent cells are thus those which have endocytosed the antibody, which then allows for endolysosomal acidification of the fluorophore. As expected, the endocytic rate is heavily concentration dependent. (B) Graphical depiction of the data presented in (A) after quantifying overall fluorescence of the treated cell populations using a fluorescent plate reader.

EXAMPLES

The invention will be illustrated by the following Examples. The Examples shall not be construed as limiting the scope of the invention.

Example 1: MUC-1 antibody elicitation strategy

Antibodies that bind to tumor-specific glycosylation variants of the mucin-1 (MUC-1) protein were generated. MUC-1 is a heavily glycosylated and unstructured protein found ubiquitously on mammalian epithelial tissues and cells, forming a mucous layer surrounding those tissues (along with several other MUC proteins). When these cells transform into cancerous cells, MUC-1 expression, function, and glycosylation are commonly perturbed. Classically, solid tumor cells overexpress and hypoglycosylate MUC-1, the combination of which leads to a loss of cell polarity and facilitates the epithelial to mesenchymal transition among many other tumorigenic phenomena. These aberrantly glycosylated and overexpressed variants of MUC-1 have been famously regarded as promising therapeutic targets, given that they represent a “tumor specific antigen.” Owing to that, several academic and pharmaceutical groups have made attempts to generate MUC-1 binding therapeutic antibodies in the past. In the majority of cases, these antibodies were conceptually designed in order to specifically drive antibodydependent tumor cell death by one of several mechanism. However, so far no MUC-1 targeting assets have successfully completed the clinical phase of evaluation, often failing to drive tumor restriction and/or displaying very poor safety profiles. These shortcomings are results of the poor quality of the antibodies that have been generated through state-of-the-art immunization tools. Several of these antibodies were even generated via immunizing mice with mixtures of intact mucins, which therefore were themselves excessively heterogeneous in terms of antigen structure. Repeated immunizations with heterogeneous and large/avid immunogens leads to the production of low-quality antibodies, especially in the contexts of specificity and affinity. These practices have successfully produced low quality antibodies that can then be classified into three basic categories in the context of binding modality: antibodies that bind to the MUC-1 glycans, antibodies that bind to the underlying MUC-1 peptide backbone, and antibodies that simultaneously bind to the peptide backbone as well as the MUC-1 glycans (Figure 1A). There are several lines of literature suggesting that some of these truncated glycans appear on a variety of other proteins all around the body during certain states of inflammation (but the absence of cancer), which brings into question the utility of antibodies that bind to the free sugar independently of peptide context. Many antibodies that do not display a sufficient level of tumor MUC-1 specificity fail in the clinic for the then to-be-expected off target toxicity effects. An example here is the Sanofi asset SAR566658, which induced a high degree of ocular toxicity events in a Phase II study of breast cancer patients, leading to the premature termination of the trial. Antibodies that bind to the sugar-less peptide backbone of MUC-1 can be of relatively higher affinity than the former category since the target is purely proteinacious, but still these antibodies have lacked the quality required to achieve clinical outcomes. A good example here is BrevaRex, an antibody that was generated already 20 years ago but has not managed to succeed in human trials. BrevaRex, despite boasting a published affinity of 43 nM, is still approximately 10,000-fold weaker than the best antibodies previously produced by the VAST platform (Triller et al., 2023).

Specificity and affinity are best achieved with glycopeptide-binding antibodies. However, it appears that the existing state-of-the-art technologies are yet to generate a high-quality glycopeptide mAb. PankoMab is the best example of this category of antibody, displaying an affinity of only 278 nM and failing to show any improvement over placebo in a large Phase II trial. However, its nature as a putative member of this category (putative because it remains difficult to assuredly claim an antibody’s mechanism of binding without a crystal structure) lends to having a promising specificity profile. An antibody with the affinity level displayed by VAST-elicited antibodies combined with the specificity profile of a true MUC-1 glycopeptide binder would be then the ideal therapeutic candidate. Thus, a series of short MUC-1 glycopeptides displaying the STn epitope bound to either a serine or threonine residue were devised. These immunogenic peptides were linked to the VAST platform to create an immunogen capable of eliciting the much sought-after antibody candidate.

Example 2: VAST-elicited antibodies can bind to glycopeptides

The VAST platform is a Trypanosoma brucei derived membranous immunogen carrier (Triller et al., 2023, WO 2020/84072, WO 2021/214043). T. brucei VSG proteins, the core of the VAST platform, are surface proteins that are themselves glycoproteins. In some cases (e.g., in the case of VSG3), these VSGs endogenously display apical O-linked glycans to the immune system. It was revealed here for the first time that the VAST platform could be used to generate glycopeptide-binding antibodies by extracting antibodies from mice that had been exposed to T. brucei that displayed VSG3 (Gkeka and Branco et al., 2023). One such antibody was assessed for its binding specificity using a flow cytometry assay. This antibody (a murine IgG) strongly binds to trypanosomes expressing VSG3, but cannot bind to trypanosomes expressing a genetic variant of VSG3 that no-longer displays the apical O-linked glycan molecule, as determined by flow cytometry via FACS Calibur. It was sought to deploy the VAST platform to generate antibodies against the glycopeptide motifs present within tumor-associated MUC-1. These epitopes are present in the variable number of tandem repeats (VNTR) domain of the protein, which is also the region targeted by several other commercial antibodies including e.g., PankomAb (Figure IB). The VAST platform could also be used to generate antibodies against the SEA domain of MUC-1 or the extracellular component of so-called MUC-1C (Figure IB).

Example 3: MUC-1 glycopeptides can be conjugated to the VAST platform

A series of glycopeptide immunogens that display the STn epitope bound to either a serine or threonine at the C-terminal end of the MUC-1 VNTR 20-mer were devised. These 20-mer peptides were elongated via the addition of the 6-residue sortase recognition motif LPSTGG (SEQ ID NO: 96). These adduct allowed for the sortase-mediated conjugation of the MUC-1 glycopeptide to the sortaggable VSG proteins that comprise the VAST platform. Importantly, the STn glycan epitope can be recognized by a series of different lectin proteins, which were used to assess VAST conjugation efficiency. Specifically, fluorescent lectins that allow for flow cytometry-based detection of cells that display the target glycans of interest were used. The Helix pomatia lectin and Sambucus nigra lectin both recognize STn, although with differing efficiencies depending on which amino acid the glycan is conjugated to. Both lectins recognized the MUC-1 labeled VAST constructs (Figure 2A), thus verifying the generation of the fully conjugated immunogens.

To verify that the soluble VSG protein had been successfully sortagged, a mass spectrometry approach was used. The sortagged VSG proteins were purified by gel filtration (e.g., as described in Triller et al., 2023) and subjected to quantitative ESI analysis. The analysis not only verified the successful sortagging reaction, but also was used to estimate the efficiency of immunogen conjugation (Table 3). These data revealed an approximate 70% sortagging efficiency for the immunogen materials, which is well above the minimum requirement needed for high quality antibody elicitation (Triller et al., 2023).

Table 3: Analysis of sortagging efficacy

The VAST materials were then used to immunize mice. An immunization schedule that is adapted for glycan immunogens (Figure 2B) was developed. The VAST system is generally designed to establish memory cell populations from immature/naive B cell precursors, and then to expand those memory cells through a recall response to promote affinity maturation. Specific glycan immunogens historically elicit only very poor affinity antibodies in only a select few responding animals. It was reasoned that the cause for this is both the rarity and low affinity of the potentially responsive naive B cells. Therefore, to promote memory establishment, a 5- inj ection paradigm that involved 2 priming injections and 3 boost injections was deployed. The primes are designed to establish memory populations via the membranous VAST immunogen. The first two boosts are designed to simultaneously establish additional memory populations via the membranous VAST immunogen, while also expand the existing memory populations via the soluble VSG protein. Finally, the 5^th injection serves to further expand all pre-existing memory populations and trigger a final round of affinity maturation.

Example 4: MUC-1 VAST immunogens elicit strong responses in the majority of immunized animals

Five C57Bl/6 mice each with either the STn-Serine MUC-1 VAST or the STn- Threonine MUC- 1 VAST were immunized. Each animal was immunized with 5 million cells-worth of VAST material (in injections 1, 2, 3, and 4) and 100 ug of soluble VSG protein (in injections 3, 4, and 5). All injections were subcutaneously delivered near the tail base. Periodically throughout the immunization schedule, small aliquots of blood were collected from each animals, including a pre-immune sample (prior to the first immunization). To assess the general quality of the immune response, serum was purified from the blood samples by centrifugation and that serum was used in ELISA assays. Streptavidin coated ELISA plates were loaded with biotinylated and shortened versions of the MUC-1 immunogen peptides. These ELISA plates were then probed with serial dilutions of the pre-immune antisera and “final bleed” antisera (collected after the 5^th immunization) from each mouse. 8 of the 10 immunized animals responded very well to the immunization (Figure 3), with 3 of 5 STn-Serine mice responding and 5 of 5 STn-Threonine mice responding. These data therefore confirmed that the VAST platform had elicited high- quality antibodies (in the form of polyclonal antiserum) that bind to MUC-1 glycopeptides. Example 5: VAST-elicited MUC-1 glycopeptide-binding antibodies display high specificities to their target immunogens

To assess the specificities of the elicited antibodies, again an ELISA system was deployed, this time using a series of different peptides as ELISA baits in addition to the cognate/immunized peptide. Antisera from animals immunized with the MUC-1 STn-Serine VAST were assayed against the cognate glycopeptide, a glycopeptide with the same amino acid sequence but with the glycan on the neighboring threonine instead, and a glycopeptide with the same amino acid sequence except that the serine-conjugated glycan was Tn instead of STn (Figure 4). These glycopeptides are some of the most similar immunogen structures that could possibly be generated. Notably, it was observed that the antisera from the 3 strongly responding animals was entirely incapable of recognizing the 2 similar glycopeptides (Figure 4). These data reveal that the antibodies present within the antisera are exquisitely specific to the STn-Serine MUC- 1 glycopeptide.

The same strategy was used to interrogate the specificities present within the antisera collected from the STn- Threonine MUC-1 VAST immunized animals. A more broad panel of specificities in these antisera was observed. Mouse (M) 1 displayed a specificity profile that did not depend on the glycan structure attached to the specific threonine, with its antisera binding equally well to STn- Threonine glycopeptide and Tn-Threonine glycopeptide (Figure 5A). However, the antisera fully discriminated against a glycopeptide with the STn glycan attached to the neighboring serine, thus still displaying a glycan-dependent binding signature (Figure 5). M2, 3, and 4 showed STn-dependent binding profiles, although the location of the STn (conjugated to either the threonine or the serine) was irrelevant (Figure 5A). Finally, M5 displayed a binding profile more similar to the animals discussed in Figure 4, with its antisera interacting only with the specific cognate/immunized glycopeptide (Figure 5A). As expected, the antisera also display specificity for the cognate glycopeptide as compared to a sugarless peptide with the same amino acid sequence, further highlighting the specificity of the antibody response (Figure 5B).

Again, a similar ELISA strategy was deployed in order to interrogate the Ig subclass that was predominating in the antisera. By using different secondary antibodies to resolve the ELISA (either targeting murine IgG or murine IgM), it was observed that the serum circulating antibodies responsible for the majority of the ELISA signal were of the IgG isotype (Figure 10). This indicates that the VAST immunization was driving an antibody response that involves class switch recombination, which is reminiscent of classical high-quality antibody development. In order to assess whether the elicited antibodies were capable of binding to MUC-1 protein present on the native surface of cancerous cells, several mouse antisera samples were used to stain tumor cells in vitro. OvCar 3 cells are a human ovarian tumor cell line well-known to display hypoglycosylated MUC-1 glyoforms. To mitigate non-specific surface binding through e.g., Fc-receptors, the cells were pre-incubated with Fc-Block. The cells were then incubated with 1 : 100 dilutions (in RPMI) of the same mouse antisera used in Figures 4 and 5, as well as antisera raised in mice immunized with a control VAST (VAST that was not sortagged to a MUC-1 peptide). Cells were then washed several times before being incubated with a 1 :250 dilution (in RPMI) of anti-mouse IgG Alexa 488 before being washed again and then analyzed on a FACS Calibur. Shown in Figure 6 are flow cytometry plots revealing that sera from mice immunized with either MUC-1 STn-SER or MUC-1 STn-THR bound the tumor cell line. The strength of detection was relatively weak, showing only a 3-4-fold shift in fluorescence relative to negative control (Figure 6). However, other studies using highly concentrated samples of purified MUC-1 monoclonal antibodies also display a similarly weak binding to tumor cells (H. Thie etal., PLoS One 2011), suggesting that these more diluted and polyclonal antisera samples contain tumor binding antibodies, albeit at low concentrations. Furthermore, optimized repetitions of this experiment including additional controls improved the quality of the data (Figure 6C), resulting in a noticeably stronger tumor cell recognition capacity than what is displayed in e.g., H. Thie et al.

Example 6: MUC-1 binding B cells can be isolated by FACS

To identify potential B cell receptor (antibody) candidate sequences, a combined FACS/single cell transcriptomics approach that has been well validated (see WO2023/161424 and Triller et al., 2023) was utilized. This approach relies on the understanding that an immunized subject/mouse will have a series of different antigen-reactive B cells within its splenocyte pool. Therefore, post immunization, spleens were extracted from immunized mice and homogenized into a cellular suspension. B cells that express surface-bound antigen-binding B cell receptors (antibodies) can then be isolated from the rest of the population by “baiting” with a fluorescent version of the MUC-1 peptide target antigen (Figure 7A). Cells that interact with the baiting moiety will stain positively for the fluorophore and can thus be identified and separated from the population. These cells are sorted each into individual wells of a 96-well plate (Figure 7A) and eventually subjected to transcriptomic-based identification of the high-quality BCR sequences.

Occasionally, certain antigen/baiting moieties will display a high degree of non-specific cell identification. In other words, a given fluorescent version of an antigen may associate with a given B cell through a BCR-independent mechanism. These mechanisms include but are not limited to non-specific interactions with the fluorophore itself and hydrophobic or proteinprotein interactions between the peptide and other components of the cell surface. To mitigate this in the context of the MUC-1 peptide baiting reagents (glycans and glycopeptides are known to display non-specific binding capacities, e.g., through lectin proteins on a given cell surface), the inventors designed minimized sTn-serine and sTn-threonine MUC-1 peptides that are flanked by a biotin moiety and a Cy5 fluorophore (Figure 7B displays the serine version). This allowed to discriminate between truly glycopeptide-binding cells and cells that e.g., bound non- specifically to the biotin moiety or internalized the peptide through some non-specific interactions with other cell surface proteins/receptors (cells that would stain as Cy5⁺ but streptavidin negative). Representative baiting data is shown in Figure 7B, where B cells (after staining and gating based on well-known B cell surface markers such as CD 19) are plotted based on their staining intensities for both the Cy5 fluorophore and a fluorescent streptavidin. A large proportion of the cells stained Cy5⁺ streptavidin', as was expected. A minor population of cells displayed the opposite phenotype, and were also discarded. Only the cells displaying both a Cy5 signal and a streptavidin signal were considered candidates for BCR sequencing (Figure 7B “Cy5⁺bio⁺”).

Example 7: MUC-1 binding B cells contain candidate MUC-1 binding antibody sequences

Candidate antibodies were identified from multiple mice immunized with the sTn-serine glycopeptide after transcriptomic-based VDJ sequencing. The combined antibody sequence candidates (from multiple mice) are depicted in circos plot form (Figure 8). Figure 8 shows the heavy and light chain VDJ gene combinations expressed by each individual potentially high- quality B cell. The light chain VJ combinations are shown on the top half of the plot, while the heavy chain VDJ combinations are shown on the bottom half. The ribbons across the plot that connect the top to the bottom delineate each individual B cell, thus revealing which light chain VJs pair with which heavy chain VDJs. Several key pattens amongst this dataset, which are used as candidate filtration prior to in vitro characterization of actual antibodies, were noticed. For example, the (*) marking in figure 8 indicates a set of three cells that each display the same VJ light chain combinations (some of which importantly originated in different mice). Additionally, the (**) mark highlights a pair of cells that are perfect clones of one another, suggesting immunization dependent expansion of this particular BCR candidate.

The cells depicted in figure 8 were replotted on FACS plots to additionally overlay information related to antigen binding capacity and somatic hypermutation count (Figure 9). Here, additional patterns allowing for high-quality hit identification were identified. For example, one cell displays 3 somatic hypermutations in its heavy chain and 6 in its light chain, relative to the germline versions of its V, D, and J genes, while other cells display fewer but still significant numbers of hypermutations. Further, several of the cells appear closer to the upper right quadrant of the plots, which thus indicates a high capacity for binding to antigen.

Based on the above-discussed patterns, the inventors identified a series of high-quality MUC- 1 -binding BCRs, which are represented in the sequence associated list.

Candidate antibodies were identified from multiple mice immunized with the sTn-threonine glycopeptide after transcriptomic-based VDJ sequencing as well. Circos plots again illustrate the enrichment of a notable handful of antibody sequences that appear more frequently than randomness would likely allow, for example antibodies incorporating heavy chain IgHVl l-2 and light chain IgKV14-126 (Figure 11A). Some of the most striking candidates are show in figure 1 IB, which illustrates the observation of two different antibody pairs that have perfectly matched Ig gene usage in both the heavy and light chain as well as very similar or identical CDR3 regions, despite originating from two different mice (Figure 11B). Since the CDR3s are not completely identical, these cells are not technically clones, which therefore suggests that this combination of V genes is utilized on a frequent basis after sTn-threonine-VAST immunization, indicating that these sequences are a likely “hit.”

The individual B cells are plotted on flowcytometry plots, illustrating the expected approximate 1 to 1 binding capacity to the two different MUC-1 bait reagents (Figure 12A). Here, the VDJ recombination profiles are overlayed onto the cells nearest to the upper right quadrant of the plot (the cells that are expected to have the highest binding capacity, which is indicative of antibody affinity). Transcriptomic profiling was used to identify the memory cells (Figure 12B)

Example 8: Monoclonal antibody expression and characterization reveals the identification of several high-quality VAST-elicited candidates

Candidate antibodies were selected for validation based on several of the discussed parameters. To produce those antibodies, the sequences of the variable regions of both the heavy and light chains of each selected antibody were synthesized. Those sequences were then cloned into expression vectors for either full length IgGl expression or antibody fragment (Fab) expression. The full length IgGl construct used here encodes a human Fc region, thus generating mousehuman chimeric antibodies. Both the IgGl and Fab expression constructs produce soluble antibodies that are secreted by the expressing cell into the media. Antibody constructs were transiently transfected into either HEK cells or CHO cells grown in suspension cultures. After 6-10 days, the cells were harvested and pelleted. The supernatant was extracted and run through a protein G-coated resin, which captures the Fc of various species of IgGl including human IgGl (Figure 13A). Purification of the IgGl from the supernatant was validated by several measures, including SDS PAGE. Successful and clean purifications with little-to-no proteolytic cleavage / degradation of the purified antibody were visualized via Coomassie staining (Figure 13B). A similar strategy was deployed for Fabs except that a nickel affinity resin was used for purification, as the Fab expression construct incorporates a HIS tag.

Several ELISA assays were used to preliminarily identify candidate binders. These ELISAs were generally successful when using IgGl versions of the discovered antibodies, however the binding of the antibodies in Fab form was far weaker. Similar observations have been observed in many cases in the literature, as it is known that the avidity effect provided by multiple linked antigen-binding regions can dramatically improve the affinity of an antibody (relative to a monovalent Fab). However, one particular antibody (candidate #135) displayed a notably strong binding capacity by ELISA even when expressed in Fab form (Figure 14). Notably, expressing PankomAb in Fab form yielded an antibody that was incapable of recognizing any MUC-1 peptides in this ELISA system (one such glycopeptide is shown in Figure 14).

All antibodies were assessed for tumor cell binding capacity, as tumor cell binding is a strong determinant for assessing whether an identified antibody can recognize “native” MUC-1 versus only the purified glycopeptide used for immunization. OvCar-3 cells (a human ovarian tumor line) display tumor-associated MUC-1, and importantly can be recognized by the IgG version of PankomAb as a positive control (Figure 15A). Several of the identified antibodies are also capable of binding to OvCar-3 cells, some of which display a stronger binding profile than PankomAb and are shown here (Figure 15A). Several other cell lines were used for these studies (including e.g, Hela cells and Colo-205 cells), each of which produced a similar binding profile to that which is shown in Figure 15 A. These studies were also supported by immunofluorescence studies as an alternative strategy. SKOV-3 cells (a different human ovarian tumor line) were used in these studies. Only a small subset of mAbs were tested in this assay system given its much lower throughput, but indeed good staining was evident (Figure 15B) for the mAbs that were tested.

Based on the above results, it was determined that several MUC-1 binders had been identified, some of which were high-quality candidates hypothetically capable of matching or beating out previously-generated competition.

Example 9: MUC-1 binders are endocytosed by tumor cells. The development of a MUC-1 therapeutic will require not only the development of an antibody with a strong binding affinity/specificity profile, but also will require the ideal choice of treatment modality. In recent years, the antibody-drug-conjugate (ADC) modality has gained traction as an attractive choice for many disease indications, including within the MUC-1 space.

Here, tumor cells are directly killed through antibody activity after a drug-bound antibody is endocytosed by the target cell, which precedes drug liberation / proteolytic cleavage from the antibody within the endolysosomal system of the cell. The drug molecule then kills the cell by one of many different mechanisms, depending on which drug molecule is selected. To assess whether the discovered antibodies could serve as templates for ADC construction, a pHrodo label was attached to several antibody candidates. pHrodo dyes are pH-sensitive molecules that only fluoresce after acidification to a certain pH. In this case, the dye chosen will not fluoresce at neutral pH, but will emit fluorescence once protonated in the acidic vacuoles of a cell after it has been endocytosed. Figure 16 illustrates one such example of this assay, wherein pHrodo- labeled antibody is endocytosed by OvCar-3 cells in an expectedly concentration-dependent manner (Figure 16A and B). It is therefore conceivable that the high-quality antibody candidates identified here are also high-quality candidates for ADC development.

Cited Literature

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Claims

1. An antibody that specifically binds to hypoglycosylated MUC1, wherein said antibody binds to an epitope formed by a carbohydrate moiety of the hypoglycosylated MUC1 and by a peptide backbone portion of the MUC1 protein.

2. The antibody of claim 1, wherein said carbohydrate moiety is sialyated N- acetylgalactosamine (GalNAc) linked to a serine or threonine residue in the peptide backbone portion by a glycosidic bond.

3. The antibody of claim 1 or 2, wherein said peptide portion comprises the amino acid sequence shown in SEQ ID NO: 95 (PGSTA).

4. The antibody of claim 3, wherein said sialyated N-acetylgalactosamine (GalNAc) is linked to the serine or threonine residue in SEQ ID NO: 95 by a glycosidic bond.

5. The antibody of any one of claims 1 to 4, wherein said hypoglycoslyated MUC1 is a cancer specific MUC1, preferably, an adenocarcinoma specific MUC1.

6. The antibody of any one of claims 1 to 5, wherein said antibody is a polyclonal or monoclonal antibody.

7. The antibody of any one of claims 1 to 6, wherein the antibody is obtainable by immunizing an animal with a VAST-bound immunogen, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

8. The antibody of any one of claim 1 to 7, wherein said antibody is obtainable by immunizing an animal using an immunization scheme having the following steps: a) two times priming using VAST-bound immunogen; b) two times boosting using VAST-bound immunogen and free immunogen; and c) one time boosting using free immunogen.

9. The antibody of claim 8, wherein the time between i) the first and second priming in step a) is about 4 weeks, ii) the second priming in step a) and the first boosting in step b) is about 4 weeks to about 6 weeks, iii) the first and the second boosting in step b) is about 4 weeks and iv) the second boosting in step b) and the boosting in step c) is about 4 weeks.

10. An antibody as defined in any one of claims 1 to 9 for use in treating and/or preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma.

11. An antibody, preferably according to any one of claims 1 to 10, comprising:

(dlO) a heavy chain as defined in (alO) or (blO), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 152, and a light chain as defined in (alO) or (blO) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(cl 1) a heavy chain as shown in SEQ ID NO: 157 and a light chain shown in SEQ ID NO: 100; or (dl 1) a heavy chain as defined in (al 1) or (bl 1), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 157, and a light chain as defined in (al 1) or (bl 1) and wherein said light chain is at least 80% identical to SEQ ID NO: 100; or

(al2) a heavy chain having a CDR1 as shown in SEQ ID NO: 162, a CDR2 as shown in SEQ ID NO: 163, a CDR3 as shown in SEQ ID NO: 164, and a light chain having a CDR1 as shown in SEQ ID NO: 101, a CDR2 as shown in SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO: 4;

(bl 2) a heavy chain having a FR1 as shown in SEQ ID NO: 249, a CDR1 as shown in SEQ ID NO: 162, an FR2 as shown in SEQ ID NO: 250, a CDR2 as shown in SEQ ID NO: 163, an FR3 as shown in SEQ ID NO: 251, a CDR3 as shown in SEQ ID NO: 164 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(cl2) a heavy chain as shown in SEQ ID NO: 161 and a light chain shown in SEQ ID NO: 104; or

(b 13) a heavy chain having a FR1 as shown in SEQ ID NO: 249, a CDR1 as shown in SEQ ID NO: 158, an FR2 as shown in SEQ ID NO: 250, a CDR2 as shown in SEQ ID NO: 159, an FR3 as shown in SEQ ID NO: 251, a CDR3 as shown in SEQ ID NO: 160 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 50, a CDR1 as shown in SEQ ID NO: 101, an FR2 as shown in SEQ ID NO: 210, a CDR2 as shown in SEQ ID NO: 3, an FR3 as shown in SEQ ID NO: 52, a CDR3 as shown in SEQ ID NO: 4 and an FR4 as shown in SEQ ID NO: 53;

(dl 3) a heavy chain as defined in (al3) or (b 13), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 165, and a light chain as defined in (al 3) or (bl 3) and wherein said light chain is at least 80% identical to SEQ ID NO: 104; or (al4) a heavy chain having a CDR1 as shown in SEQ ID NO: 168, a CDR2 as shown in SEQ ID NO: 169, a CDR3 as shown in SEQ ID NO: 170, and a light chain having a CDR1 as shown in SEQ ID NO: 106, a CDR2 as shown in SEQ ID NO: 107, a CDR3 as shown in SEQ ID NO: 108;

(al 5) a heavy chain having a CDR1 as shown in SEQ ID NO: 283, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 284, and a light chain having a CDR1 as shown in SEQ ID NO: 110, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 112;

(b 15) a heavy chain having a FR1 as shown in SEQ ID NO: 256, a CDR1 as shown in SEQ ID NO: 283, an FR2 as shown in SEQ ID NO: 257, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 284 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 216, a CDR1 as shown in SEQ ID NO: 110, an FR2 as shown in SEQ ID NO: 217, a CDR2 as shown in SEQ ID NO: 111, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 112 and an FR4 as shown in SEQ ID NO: 61;

(dl 5) a heavy chain as defined in (al5) or (b 15), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 282, and a light chain as defined in (al 5) or (bl 5) and wherein said light chain is at least 80% identical to SEQ ID NO: 109; or

(al6) a heavy chain having a CDR1 as shown in SEQ ID NO: 283, a CDR2 as shown in SEQ ID NO: 150, a CDR3 as shown in SEQ ID NO: 172, and a light chain having a CDR1 as shown in SEQ ID NO: 110, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 112; (b 16) a heavy chain having a FR1 as shown in SEQ ID NO: 256, a CDR1 as shown in SEQ ID NO: 283, an FR2 as shown in SEQ ID NO: 257, a CDR2 as shown in SEQ ID NO: 150, an FR3 as shown in SEQ ID NO: 244, a CDR3 as shown in SEQ ID NO: 172 and an FR4 as shown in SEQ ID NO: 72, and a light chain having an FR1 as shown in SEQ ID NO: 216, a CDR1 as shown in SEQ ID NO: 110, an FR2 as shown in SEQ ID NO: 217, a CDR2 as shown in SEQ ID NO: 111, an FR3 as shown in SEQ ID NO: 218, a CDR3 as shown in SEQ ID NO: 112 and an FR4 as shown in SEQ ID NO: 61;

(dl7) a heavy chain as defined in (al7) or (bl7), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 173, and a light chain as defined in (al 7) or (bl 7) and wherein said light chain is at least 80% identical to SEQ ID NO: 113; or

(al8) a heavy chain having a CDR1 as shown in SEQ ID NO: 155, a CDR2 as shown in SEQ ID NO: 177, a CDR3 as shown in SEQ ID NO: 178, and a light chain having a CDR1 as shown in SEQ ID NO: 118, a CDR2 as shown in SEQ ID NO: 115, a CDR3 as shown in SEQ ID NO: 108;

(dl 8) a heavy chain as defined in (al8) or (b 18), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 176, and a light chain as defined in (al 8) or (bl 8) and wherein said light chain is at least 80% identical to SEQ ID NO: 117; or

(al 9) a heavy chain having a CDR1 as shown in SEQ ID NO: 180, a CDR2 as shown in SEQ ID NO: 181, a CDR3 as shown in SEQ ID NO: 182, and a light chain having a CDR1 as shown in SEQ ID NO: 120, a CDR2 as shown in SEQ ID NO: 121, a CDR3 as shown in SEQ ID NO: 122;

(dl9) a heavy chain as defined in (al9) or (bl9), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 179, and a light chain as defined in (al 9) or (bl 9) and wherein said light chain is at least 80% identical to SEQ ID NO: 119; or

226, a CDR1 as shown in SEQ ID NO: 124, an FR2 as shown in SEQ ID NO: 227, a CDR2 as shown in SEQ ID NO: 125, an FR3 as shown in SEQ ID NO: 228, a CDR3 as shown in SEQ ID NO: 126 and an FR4 as shown in SEQ ID NO: 229; (c20) a heavy chain as shown in SEQ ID NO: 183 and a light chain shown in SEQ ID NO: 123; or

(d20) a heavy chain as defined in (a20) or (b20), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 183 and a light chain as defined in (a20) or (b20) and wherein said light chain is at least 80% identical to SEQ ID NO: 123 or

(c22) a heavy chain as shown in SEQ ID NO: 189 and a light chain shown in SEQ ID NO: 127; or (d22) a heavy chain as defined in (a22) or (b22), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 189, and a light chain as defined in (a22) or (b22) and wherein said light chain is at least 80% identical to SEQ ID NO: 127; or

(d24) a heavy chain as defined in (a24) or (b24), and wherein said heavy chain is at least 80% identical to SEQ ID NO: 195, and a light chain as defined in (a24) or (b24) and wherein said light chain is at least 80% identical to SEQ ID NO: 131; or (a25) a heavy chain having a CDR1 as shown in SEQ ID NO: 198, a CDR2 as shown in SEQ ID NO: 199, a CDR3 as shown in SEQ ID NO: 200, and a light chain having a CDR1 as shown in SEQ ID NO: 136, a CDR2 as shown in SEQ ID NO: 111, a CDR3 as shown in SEQ ID NO: 137;

(a27) a heavy chain having a CDR1 as shown in SEQ ID NO: 202, a CDR2 as shown in SEQ ID NO: 203, a CDR3 as shown in SEQ ID NO: 204, and a light chain having a CDR1 as shown in SEQ ID NO: 139, a CDR2 as shown in SEQ ID NO: 140, a CDR3 as shown in SEQ ID NO: 143; (b27) a heavy chain having a FR1 as shown in SEQ ID NO: 276, a CDR1 as shown in SEQ ID NO: 202, an FR2 as shown in SEQ ID NO: 277, a CDR2 as shown in SEQ ID NO: 203, an FR3 as shown in SEQ ID NO: 278, a CDR3 as shown in SEQ ID NO: 204 and an FR4 as shown in SEQ ID NO: 82, and a light chain having an FR1 as shown in SEQ ID NO: 236, a CDR1 as shown in SEQ ID NO: 139, an FR2 as shown in SEQ ID NO: 237, a CDR2 as shown in SEQ ID NO: 140, an FR3 as shown in SEQ ID NO: 238, a CDR3 as shown in SEQ ID NO: 143 and an FR4 as shown in SEQ ID NO: 61;

12. A VAST-bound immunogen for use as a vaccine, wherein said VAST-bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP) wherein sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

13. The VAST-bound immunogen for use of claim 12, wherein said vaccine is suitable for preventing cancer associated with aberrant cancer specific MUC1 expression and, preferably, adenocarcinoma.

14. A method for manufacturing an antibody that specifically binds to hypoglycosylated MUC1, said method comprising the step of obtaining the antibody from a sample of an animal which has been immunized with a VAST-bound immunogen, wherein said VAST- bound immunogen comprises a peptide having an amino acid sequence as shown in SEQ ID NO: 92 (PAHGVTSAPDTRPAPGSTAP), wherein sialyated N-acetylgalactosamine (GalNAc) is linked to the serine residue at position 17 or threonine residue at position 18 in SEQ ID NO: 92 by a glycosidic bond.

15. The method of claim 14, wherein said animal has been immunized using an immunization scheme having the following steps: a) two times priming using VAST-bound immunogen; b) two times boosting using VAST-bound immunogen and free immunogen; and c) one time boosting using free immunogen.

16. The method of claim 15, wherein the time between i) the first and second priming in step a) is about 4 weeks, ii) the second priming in step a) and the first boosting in step b) is about 4 weeks to about 6 weeks, iii) the first and the second boosting in step b) is about 4 weeks and iv) the second boosting in step b) and the boosting in step c) is about 4 weeks.