Novel peptide-based compounds for use in the prevention, treatment and / or detection of cancer - Patents.com

Abstract

The present invention relates to a compound comprising a first polypeptide sequence comprising at least three different amino acid sequences selected from the group consisting of -GFTFSDYW (SEQ ID NO: 1), IRLKSNNYAA (SEQ ID NO: 2) and TFGNSFAY (SEQ ID NO: 3), and a second polypeptide sequence comprising at least three different amino acid sequences selected from the group consisting of -TGAVTTNNY (SEQ ID NO: 4), GTN (SEQ ID NO: 5) and ALWYSNHWV (SEQ ID NO: 6), or a variant of any one of said amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 6 having a different amino acid at one position, The present invention relates to compounds or variants that are capable of binding to tumor-associated mucin-1 (TA-MUC1). [Selected Figure] Figure 1

Description

[Technical field]

[0001] The present invention relates to novel compounds capable of binding to mammalian tumor-associated mucin-1 (TA-MUC1) that can be found in MUC1-expressing tumor tissues. The compounds of the present invention are suitable for treating, preventing or detecting cancers such as pancreatic cancer, prostate cancer, breast cancer, B-cell leukemia, multiple myeloma, colon cancer and ovarian cancer. Thus, the compounds of the present invention allow for the differentiation of TA-MUC1 found in epithelial tumor cells from MUC-1 found in healthy epithelial cells. Thus, the compounds of the present invention are suitable as tumor targeting agents for use in therapeutic and diagnostic applications. [Background technology]

[0002] MUC1 is a highly glycosylated protein expressed in many epithelial tissues and can be found, for example, in breast, colon, prostate, pancreatic tissues, and ovaries (MA Hollingsworth, BJ Swanson, Nat. Rev. Cancer, 2004, 4, 45-60). The extracellular portion of MUC1 contains a domain consisting of 20-100 repeated copies of the sequence PAHGVTSAPDTRPAPGSTAP, including five O-glycosylation sites at serine or threonine. Normally, MUC1 carries long glycans, which causes the protein to adopt an extended conformation. Due to the dense glycosylation, the peptide backbone is inaccessible to the immune system. The glycans of TA-MUC1 are much shorter and often sialylated early (J. Arklie, J. Taylor-Papadimitrious et al., Int. J. Cancer, 1981, 28, 23-29; I. Brockhausen et al., Eur. J. Biochem., 1995, 233, 607-617; F.-G. Hanisch et al., Glycoconjugate J., 1990, 7, 525-543; N. Domenech et al., J. Immunol., 1995, 155, 4766-4774).

[0003] Application of such glycoproteins in the course of immunotherapy may induce undesirable severe autoimmune reactions against normal MUC1. Pure TA-MUC1-glycopeptide antigens cannot be isolated from tumor cells and must be obtained by total chemical synthesis (D. Samet al., Nature, 1997, 389, 587-559; B. Liebe, H. Kunz, Angew. Chem. Int. Ed. Engl., 1997, 36, 618-621; Angew. Chem., 1997, 109, 629-631).

[0004] O-glycans are incompletely processed in cancer cells, resulting in the expression of pancreatic cancer carbohydrate antigens Tn (GalNAccd-O-Ser / Thr), STn (NeuAca2-6GalNAca1-O-Ser / Thr), and T (Gal31-3GalNAca1-0-Ser / Thr). MUC1 expressed by breast cancer cells carries the short cancer-associated Tn, STn, and T antigens commonly found in normal cells, as well as mono- and disialyl core 1 structures (ST, NeuAca2-3Gal31-3[NeuAca2-6]+ / -GalNAca1-0-Ser / Thr). In contrast, MUC1 expressed by normal breast epithelial cells generally carries branched core 2 O-glycans with lactosamine extensions (Gal31-3[GlcNAc31-6]GalNAca1-0-Ser / Thr).

[0005] Cell membrane-bound MUC1 has long been considered a primary target for immunotherapeutic intervention. However, stimulation of effective cellular or humoral immune responses against cancer-associated forms of MUC1 has not been achieved in patients or transgenic animals expressing the human MUC1 gene (using defined immunogens, as opposed to cell-based therapies). Active specific immunotherapy strategies based on peptide / protein immunogens have so far been limited to non-glycosylated MUC1 tandem repeat peptides of different lengths, conjugated to different carriers, or administered with adjuvants. These strategies have generally failed to generate an effective immune response against MUC1 expressed by cancer cells in hosts where mucin is expressed as a self-antigen.

[0006] The N-terminal subunit of MUC1, which contains a variable number of tandem repeats with a high percentage of serine and threonine residues that are modified by O-glycosylation, extends beyond the cellular glycocalyx and is tethered to the cell surface via non-covalent binding to the transmembrane C-terminal subunit of MUC1 (Merlo, 1989). The C-terminus consists of a 58 amino acid extracellular domain, a 28 amino acid transmembrane domain, and a 72 amino acid cytoplasmic domain that interacts with a variety of signaling molecules (Kufe, 2008).

[0007] In the past, numerous monoclonal antibodies (mAbs) have been produced to purify MUC1, synthetic peptides derived from MUC1, and glycopeptides. The epitopes of these mAbs have traditionally been defined by scanning short overlapping peptides, with most mAbs defining epitopes within the highly O-glycosylated mucin tandem repeat domain. One large group of mAbs has been generated against human milk fat globules (HMFG), such as HMFG1, 115D8, and SM3, most of which react with epitopes within the PDTR region of the MUC1 tandem repeats, which are considered immunodominant peptide epitopes. Most MUC1 antibodies react with the peptide backbone, but in many cases binding is modulated by the presence of glycans. In tumor cells, the peptide backbone of MUC1 appears to be accessible to the immune system but is hindered by residual aberrant glycosylation (I. Brockhausen et al., Eur. J. Biochem., 1995, 233, 607-617; SJ Gendle et al., J. Biol. Chem., 1990, 265, 15286-15293).

[0008] TA-MUC1 differs from MUC1 expressed in normal cells in terms of its biochemical characteristics, cellular distribution, and function (Nathetal., Trends in Molecular Medicine, June 2014, Vol. 20, Issue 6, p. 332-342). In cancer cells, MUC1 participates in intracellular signaling pathways and regulates the expression of its target genes at both transcriptional and post-transcriptional levels.

[0009] However, to exploit this structural difference for immunotherapeutic and diagnostic purposes, the immune system needs to be specifically activated against these TA-MUC1 structures. This can only be achieved with complex synthetic glycopeptides that mimic antigens that selectively display unique components of tumor-associated MUC1. Because tumor cell transferase activities are highly diverse in nature, both normal and tumor-associated glycans can be found on a single MUC1 molecule, allowing the generation of novel compounds that are suitable for specifically targeting TA-MUC1 found on epithelial tumor cells.

[0010] US Patent Application Publication No. 2016 / 0176975 describes a method for inducing a cancer-specific immune response against TA-MUC1 using immunogenic glycopeptides containing a GSTA motif that is O-glycosylated at the S / T residues (STN / TN antigen). In particular, monoclonal antibodies such as mAbs 5E5, 5E10, 1B9, 2D9 are compared with previously reported known mAbs such as SM3, BW835.

[0011] US Patent No. 9,546,201 describes peptides from specific regions of the MUC1 cytoplasmic domain that have been shown to inhibit MUC1 oligomerization and nuclear translocation, and methods for preventing tumor cell growth by inducing tumor cell apoptosis and tumor tissue necrosis in vivo. The oligomerization inhibitor peptide comprises 20 or fewer consecutive residues of the amino acid sequence CQCRRKN ​​and a cellular delivery domain such as poly-DR that covers the amino-terminal cysteine ​​of the peptide. The peptide can be applied to any cell that expresses MUC1, more specifically to cells that overexpress MUC1.

[0012] WO 2013 / 185552 discloses chimeric antigen receptors for treating malignant tumors and viral infections. China Patent Application Publication No. 107184602 and China Patent Application Publication No. 103483453 disclose chimeric antigen receptors for use in treating tumors. WO 2013 / 103658 discloses peptides containing epitopes from the human tumor-associated (TA) antigen mucin 1 (MUC1) for use in preventing or treating cancer by vaccination.

[0013] However, it remains desirable to obtain compounds that can specifically distinguish between normal MUC1 found in healthy epithelial cells and TA-MUC1 found in tumor tissues, thereby providing a high rate of tumor-selective recognition in order to be suitable / useful as active agents for clinical applications in the treatment, prevention, and / or diagnosis of tumor entities expressing TA-MUC1. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] US Patent Application Publication No. 2016 / 0176975 [Patent Document 2] U.S. Patent No. 9,546,201 [Patent Document 3] International Publication No. 2013 / 185552 [Patent Document 4] China Patent Application Publication No. 107184602 [Patent Document 5] China Patent Publication No. 103483453 [Patent Document 6] International Publication No. 2013 / 103658 [Non-patent literature]

[0015] [Non-Patent Document 1] MAHollingsworth,BJSwanson,Nat.Rev.Cancer,2004,4,45-60 [Non-licensed document 2] J.Arklie,J.Taylor-Papadimitrious et al.,Int.J.Cancer,1981,28,23-29 [Non-licensed document 3] I.Brockhausen et al.,Eur.J.Biochem.,1995,233,607-617

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[0016] It is therefore an object of the present invention to provide alternative compounds which allow for the discrimination between TA-MUC1 found in healthy epithelial cells and normal MUC1 and which are suitable for the treatment, prevention and diagnosis of cancer, including tumor cells which specifically express tumor-associated MUC1. [Means for solving the problem]

[0017] This object is solved by a peptide-based compound according to claim 1. Preferred embodiments of the invention are part of the dependent claims. [Brief description of the drawings]

[0018] [Figure 1] Histological staining of human breast cancer biopsies for TA-MUC1 using α-hu-TA-MUC1. A: A collection of breast cancer tissue sections from 142 patients was examined for TA-MUC1 staining specifically for the diagnosis of breast cancer. Human breast cancer cells (MCF-7), which strongly express TA-MUC1, served as a positive control, and paraffin sections of healthy glandular tissue and paraffin sections of HR+ breast tumors were examined. Representative examples of 142 breast cancer tissue sections and 10 breast glandular tissue sections are shown. B: Correlation of TA-MUC1 expression with metastasis-free (MFS) and recurrence-free (RFS) patient survival. Staining intensity (0=negative, 1=weak, 2=moderate, 3=strong) was multiplied by the percentage of MUC1-positive tumor cells (0=0%, 1=1-10%, 2=11-50%, 3=51-80%, 4=81-100%). [Diagram 2]Immunohistochemical staining of cryogenic tissue sections of mammary glands and mammary tumors for hu-TA-MUC1 with α-hu-TA-MUC1. Mammary tumors isolated from PyMT-tg mice not expressing hu-TA-MUC1, mammary tissues isolated from tumor-free MUC1-tg mice expressing huMUC1, and mammary tumors isolated from PyMTxhuMUC1 mice expressing hu-TA-MUC1 were stained with α-hu-TA-MUC1 labeled with AF647N far-red fluorescent dye (5 pg / ml). [Diagram 3] Figure 1: PyMTxhuMUC1 mouse tumor cells from primary tumor cell lines express human MUC1. PyMTxhuMUC1 and PyMT tumors were isolated and digested ex vivo to obtain single cell tumor cell cultures. A: Human MUC1 expression of generated mouse breast tumor cells was determined via qRT-PCR in comparison to a human MUC1 expressing cell line (T47D, positive control). B16F10 melanoma cells served as a negative control. B: FACS analysis using α-hu-TA-MUC1 mAb confirmed hu-TA-MUC1 protein expression of primary PyMTxhuMUC1-derived breast tumor cell lines. [Figure 4]Figure showing tumor size reduction by prophylactic immunization against hu-TA-MUC1. PyMT-tg mice (n = 5) and PyMTxhuMUC1-tg mice (n = 5) were immunized three times every 14 days with 10 pg of hu-TA-MUC1-glycopeptide-TTox vaccine. Untreated PyMT-tg mice (n = 5) and untreated PyMTxhuMUC1-tg mice (n = 5) were used as controls. A: 20-week-old mice were sacrificed and tumor size was measured. The average size of all tumors in one mouse in mm2 was calculated. Unpaired two-sided t-test. **<P0.01, ****<P0.0001. B: Half of the maximum titer of IgG antibody was determined by ELISA after the third immunization. C: Half of the maximum titer of different Ig subtypes was determined by ELISA after the third immunization. Microtiter plates were coated with hu-TA-MUC1-glycopeptide-BSA conjugate corresponding to the TA-MUC1-glycopeptide part of the vaccine. Only hu-TA-MUC1-binding antibodies were quantified. Unpaired two-sided t-test. ****<P0.0001. [Diagram 5] Figure showing the extension of survival time of breast tumor-bearing mice by prophylactic immunization against hu-TA-MUC1. PyMTxhuMUC1 mice (n = 5) were immunized three times at 14-day intervals with 10 pg of hu-TA-MUC1-glycopeptide-TTox vaccine. Untreated PyMTxhuMUC1 mice (n = 5) were used as control mice. A: The health status and behavior of breast tumor-bearing mice were observed. Mice were sacrificed when the tumor size reached approximately 200 mm2 and / or when food intake and physiological agility were adversely affected. The number of days of survival after birth was displayed. Log-rank Mantel-Cox test; ***p<0.001. B: Half of the maximum titer of IgG antibody was determined by ELISA after the third immunization. C: Half of the maximum titer of different IgG subtype antibodies was determined by ELISA after the third immunization. Microtiter plates were coated with hu-TA-MUC1-glycopeptide-BSA conjugate. Only hu-TA-MUC1-binding antibodies were quantified.

Mode for Carrying Out the Invention

[0019] The compounds of the present invention are obtained by immunization with specific MUC1-derived glycopeptides that mimic the structure of TA-MUC1 on epithelial tumor cells and contain one or more polypeptide sequences that contain the specific binding site required for binding to mammalian TA-MUC1. Surprisingly, the binding site identified in the present invention is specific for TA-MUC1 but not for normal MUC1 found in healthy epithelial tissue. Thus, the compounds of the present invention are suitable for distinguishing between tumor cells expressing TA-MUC1 and healthy cells expressing normal MUC1. The binding site of the compounds of the present invention was identified by using an immunogenic vaccine that resulted in the generation of a TA-MUC1-specific mouse mAb. The amino acid sequence of the binding site and binding region of one mAb has been identified and is defined herein as SEQ ID NO:1 to SEQ ID NO:6.

[0020] A polypeptide-based compound that includes a binding site specific for binding to mammalian TA-MUC1, preferably human TA-MUC1 (hu-TA-MUC1), - a first polypeptide sequence comprising at least three different amino acid sequences selected from the group consisting of GFTFSDYW (SEQ ID NO: 1), IRLKSNNYAA (SEQ ID NO: 2) and TFGNSFAY (SEQ ID NO: 3); - a second polypeptide sequence comprising at least three different amino acid sequences selected from the group consisting of TGAVTTNNY (SEQ ID NO: 4), GTN (SEQ ID NO: 5) and ALWYSNHWV (SEQ ID NO: 6), or a variant of any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6 has a different amino acid at one or more positions, The compound or variant is capable of binding to TA-MUC1.

[0021] Thus, the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:3 of the first polypeptide sequence carrying a binding site for binding to TA-MUC1 can be arranged in the following form: GFTFSDYW (SEQ ID NO: 1)-IRLKSNNYAA (SEQ ID NO: 2)-TFGNSFAY (SEQ ID NO: 3), GFTFSDYW (SEQ ID NO: 1)-TFGNSFAY (SEQ ID NO: 3)-IRLKSNNYAA (SEQ ID NO: 2), IRLKSNNYAA (SEQ ID NO: 2)-TFGNSFAY (SEQ ID NO: 3)-GFTFSDYW (SEQ ID NO: 1), IRLKSNNYAA (SEQ ID NO: 2)-GFTFSDYW (SEQ ID NO: 1)-TFGNSFAY (SEQ ID NO: 3), TFGNSFAY (SEQ ID NO: 3)-GFTFSDYW (SEQ ID NO: 1)-IRLKSNNYAA (SEQ ID NO: 2), TFGNSFAY (sequence number 3)-IRLKSNNYAA (sequence number 2)-GFTFSDYW (sequence number 1).

[0022] Similarly, the amino acid sequences of SEQ ID NO:4 to SEQ ID NO:6 of the second polypeptide sequence carrying an additional binding site required for binding to mammalian TA-MUC1, preferably hu-TA-MUC1, can be arranged in the following form: TGAVTTNNY (SEQ ID NO: 4)-GTN (SEQ ID NO: 5)-ALWYSNHWV (SEQ ID NO: 6), TGAVTTNNY (SEQ ID NO: 4)-ALWYSNHWV (SEQ ID NO: 6)-GTN (SEQ ID NO: 5), GTN (SEQ ID NO: 5)-TGAVTTNNY (SEQ ID NO: 4)-ALWYSNHWV (SEQ ID NO: 6), GTN (SEQ ID NO: 5)-ALWYSNHWV (SEQ ID NO: 6)-TGAVTTNNY (SEQ ID NO: 4), ALWYSNHWV (SEQ ID NO: 6)-TGAVTTNNY (SEQ ID NO: 4)-GTN (SEQ ID NO: 5), ALWYSNHWV (sequence number 6)-GTN (sequence number 5)-TGAVTTNNY (sequence number 4).

[0023] The present invention includes variants of any one of the amino acid sequences defined in SEQ ID NO:1-6 that contain one or more amino acid substitutions at one or more positions within the amino acid sequence. However, the present invention is directed only to variants that are capable of binding to mammalian TA-MUC1, preferably hu-TA-MUC1, and thus retain the binding specificity of the compounds of the invention. Variants may also include amino acid sequences that are homologous to SEQ ID NO:1-6 found in the corresponding binding regions of different mammalian species, such as human homologous sequences.

[0024] Having a different amino acid at one or more positions within any of the amino acid sequences of SEQ ID NO:1-6 means that more than one, two, three or more amino acids may differ from the sequence outlined in SEQ ID NO:1-2, either by substitution, addition, deletion or modification. Preferably, in such variants, one, two, three or four amino acids in a given amino acid sequence are replaced by one or more alternative amino acids. In a preferred embodiment, no more than three, preferably no more than two amino acids are replaced by another amino acid.

[0025] The variant according to the present invention may comprise an amino acid sequence having at least 70% positive amino acid identity, more preferably at least 90% positive amino acid identity, for example 91% to 99% positive amino acid identity, with any one of the selected amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6. A positive amino acid identity is defined as an amino acid residue having similar physical, biological and / or chemical properties present at the same position in the two compared sequences. Preferred positive amino acid identity that can be used for substitution are K to R, E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S, and Q to R. The peptide or variant of the present invention should have the ability to specifically bind to mammalian TA-MUC1 in tumor cells but not to normal MUC1 found in normal cells.

[0026] The compounds or variants according to the invention may also contain other chemical moieties, such as phosphoryl, sulfur, acetyl, or glycosyl moieties. Thus, a given amino acid sequence may be modified, for example, by addition, deletion, substitution, or chemical modification of one or more amino acid residues. Both L-amino acids and D-amino acids may be used for such modifications. Possible chemical modifications may include sugar or ester derivatives, such as methyl or acetyl esters, or polyethylene glycol modifications. Furthermore, the amine groups of peptides may be converted to amides, including fatty acids. The peptides or variants of the invention may also be modified by biotin at their C-terminus or by His-tag at their N-terminus.

[0027] According to the present invention, a variant of an amino acid sequence may contain one or more conservative amino acid substitutions that are independent of each other, (i) at least one glycine (Gly) of said variant is replaced with an amino acid selected from the group of amino acids consisting of Ala, Val, Leu, and lie; (ii) at least one alanine (Ala) of said variant is replaced with an amino acid selected from the group of amino acids consisting of Gly, Val, and Leu; (iii) at least one valine (Val) of said variant is replaced with an amino acid selected from the group of amino acids consisting of Gly, Ala, Leu, and lie; (iv) at least one leucine (Leu) of said variant is replaced with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and Leu; (v) at least one isoleucine (lie) of this variant is replaced with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and Leu; (vi) at least one aspartic acid (Asp) of the variant is replaced with an amino acid selected from the group of amino acids consisting of Glu, Asn, and Gin; (vii) at least one asparagine (Asn) of the variant is replaced with an amino acid selected from the group of amino acids consisting of Asp, Glu, and Gin; (viii) at least one glutamine (Gin) of the variant is replaced with an amino acid selected from the group of amino acids consisting of Asp, Glu, and Asn; (ix) at least one phenylalanine (Phe) of the variant is replaced with an amino acid selected from the group of amino acids consisting of Tyr, Trp, His, Pro, preferably selected from the group of amino acids consisting of Tyr and Trp; (ix) at least one tyrosine (Tyr) of the variant is replaced with an amino acid selected from the group of amino acids consisting of Phe, Trp, His, Pro, preferably selected from the group of amino acids consisting of Phe and Trp; (xi) at least one arginine (Arg) of this variant is replaced with an amino acid selected from the group of amino acids consisting of Lys and His; (xii) at least one lysine (Lys) of this variant is replaced with an amino acid selected from the group of amino acids consisting of Arg and His; (xiii) at least one proline (Pro) of the variant is replaced with an amino acid selected from the group of amino acids consisting of Phe, Tyr, Trp, and His; (xiv) At least one cysteine ​​(Cys) of the variant is substituted with an amino acid selected from the group of amino acids consisting of Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, and Tyr.

[0028] It therefore follows from the above that the same variant of a compound of the invention may contain multiple conservative or homologous amino acid substitutions from multiple groups of conserved amino acids defined herein above.

[0029] Conservative amino acid substitutions can be introduced at any position of the polypeptide sequence of the present invention. However, it may be desirable to introduce non-conservative substitutions, particularly, but not limited to, at one or more positions. Amino acid substitutions can be made based on their hydrophobicity and hydrophilicity values, as well as the relative similarity of the amino acid side-chain substituents, such as size and charge.

[0030] Conserved amino acid groups, according to their known chemical properties, are preferably the following: (i) A, G; (ii) Q, N, S, T; (iii) E, D; (iv) Q, N, S, T; (v) H, K, R; (vi) L, P, I, V, M, F, Y, W.

[0031] In the context of the present invention, the standard one-letter code as well as the standard three-letter code of amino acid residues applies. The abbreviations of amino acids comply with the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur.J.Biochem,1984,vol.184,pp9-37. Throughout the description and claims, either the three-letter code or the one-letter code of natural amino acids is used. When the L- or D-form is not specified, it is to be understood that the amino acid in question has the natural L-form (see Pure&Appl.Chem.Vol.56(5)pp595-624(1984)) or the D-form. Thus, the peptide formed may be composed of sequences of L-, D-form amino acids or mixed L- and D-forms.

[0032] The C-terminal amino acid of a peptide is usually present as a free carboxylic acid, which may also be designated as "-OH". However, the C-terminal amino acid of a peptide for use according to the present invention may also be an amidated derivative, designated as "-NH-2". Unless otherwise noted, the N-terminal amino acid of a polypeptide contains a free amino group, which may also be designated as "H-". A peptide, fragment or variant thereof according to the present invention may also contain one or more unnatural amino acids or modified natural amino acids.

[0033] In a preferred embodiment of the invention, the different amino acid sequences comprising binding sites for binding mammalian TA-MUC1, preferably hu-TA-MUC1, are arranged in the compound of the invention as follows: a first polypeptide sequence comprising the structure A1-x-A2-y-A3, A1 comprises the amino acid sequence set forth in SEQ ID NO:1, A2 comprises the amino acid sequence set forth in SEQ ID NO:2, A3 comprises the amino acid sequence set forth in SEQ ID NO:3, where x, y are 0 or more linking amino acids; a second polypeptide sequence having the structure A4-x-A5-y-A6, A4 comprises the amino acid sequence set forth in SEQ ID NO:4, A5 comprises the amino acid sequence set forth in SEQ ID NO:5, A6 comprises the amino acid sequence set forth in SEQ ID NO:6, where x, y are 0 or more linking amino acids; or a variant of any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6 having a different amino acid at one or more positions. According to the invention, the compound or variant is capable of binding to TA-MUC1.

[0034] The linking amino acids x or y can be random amino acids of various lengths. The typical length of x or y is 15-70 amino acids, but preferably 75 amino acids or less. When x or y is 0, this means that there are no linking amino acids to connect the TA-MUC1 binding sites defined in the amino acid sequences of SEQ ID NO:1-SEQ ID NO:6.

[0035] In a preferred embodiment of the first polypeptide sequence, x represents the amino acid sequence MNWVRQSPEKGLEWVAD, or variants of that amino acid sequence have different amino acids at one or more positions. In a preferred embodiment of the first polypeptide sequence, y represents the amino acid sequence HYAESVKGRFTVSRDDSKSSVYLQMNNLRTEDTGFYYC, or variants of that amino acid sequence have different amino acids at one or more positions.

[0036] In a preferred embodiment of the second polypeptide sequence, x represents the amino acid sequence ANWVQEKPDHLFTGLIG, or variants of that amino acid sequence have different amino acids at one or more positions. In a preferred embodiment of the second polypeptide sequence, y represents the amino acid sequence NRGPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC, or variants of that amino acid sequence have different amino acids at one or more positions.

[0037] All variants of the present invention that contain homologous binding sequences found in other species shall have the biological activity of recognizing and binding to mammalian TA-MUC1, preferably hu-TA-MUC1.

[0038] In a preferred embodiment, the first polypeptide sequence and the at least one second polypeptide sequence are covalently linked by an additional linker.

[0039] Preferably, the linker comprises one or more amino acids of 1 to 25 amino acids selected from the group consisting of S and G. The linker is therefore rich in the amino acids S and G, for example in the form SGGGGS. However, the linker may also comprise one or more different amino acids within the sequence or at its termini.

[0040] Preferably, the compound of the invention is a protein, an antibody, a single chain fragment variable (scFv) or a hybrid fusion protein. The hybrid fusion protein may be a compound of the invention linked to another protein, such as an enzyme, an antibody (fragment) or a cytokine.

[0041] In a preferred embodiment, the compound of the invention is 89 Zr or 117 It can be labeled with Lu. 89 Zr is suitable for analytical imaging to detect cancer tissues expressing TA-MUC1. 117 Lu is suitable as a beta radiator for tumor treatment because this compound can destroy tumor cells while sparing the surrounding healthy tissue.

[0042] To purify the compound of the present invention, the compound can be bound to histidine tag or biotin. To verify the binding of the compound of the present invention to TA-MUC1, streptavidin tag or luminescent or fluorescent dye can be used. The compound can also be bound to an enzyme such as alkaline phosphatase to detect tumor cells in the analyzed tissue. To increase the specificity of the binding of the compound of the present invention, nanoparticles can be used for tumor targeting.

[0043] In a preferred embodiment, the compounds of the invention can be conjugated to CD3 or a derivative of CD3mAb, for example in the form of a CD3-scFv, to bind CD3+ T cells to TA-MUC1 expressing tumor cells. Conjugation of CD3 to the compounds of the invention can significantly improve tumor cell lysis, for example, by cytotoxic T cells.

[0044] Preferably, the compound of the present invention has a molecular weight of 15 to 100 kDa, more preferably 25 to 50 kDa. Small compounds are preferred because they have less steric hindrance and therefore do not interfere with binding to TA-MUC1.

[0045] Preferably, the compound of the invention is an antibody or an antibody fragment such as an scFv, and the first polypeptide sequence is a portion of the variable region of the heavy chain (VH) of the antibody and the second polypeptide sequence is a portion of the variable region of the light chain (VL) of the antibody or antibody fragment.

[0046] In a preferred proteinaceous compound of the present invention, the first polypeptide sequence comprises the amino acid sequence set forth in SEQ ID NO:7 and the second polypeptide sequence comprises the amino acid sequence set forth in SEQ ID NO:8, or a variant of either one of the amino acid sequences of SEQ ID NO:7 or SEQ ID NO:8 having a different amino acid at one or more positions, which variant is capable of binding to mammalian TA-MUC1, preferably hu-TA-MUC1.

[0047] Preferably, the first and second polypeptide sequences are portions of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9 or a variant of the amino acid sequence of SEQ ID NO:9 having a different amino acid at one or more positions, which variant is capable of binding to tumor-associated MUC1.

[0048] As shown in the following examples, a protein composed of a first polypeptide sequence comprising the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:3 and a second polypeptide sequence comprising the amino acid sequences of SEQ ID NO:4 to SEQ ID NO:6 is suitable for recognition of surface-bound TA-MUC1 in tumor tissues, but not in healthy tissues expressing MUC1. Thus, the compounds of the present invention are suitable for distinguishing cancer tissues from normal tissues expressing differently glycosylated MUC1 subtypes. The following examples also illustrate that prophylactic immunization against hu-TA-MUC1 by antitumor vaccines applied to induce the compounds of the present invention can reduce tumor size. Thus, the compounds of the present invention are suitable for the prevention or treatment of cancers characterized by expressing abnormally glycosylated TA-MUC1.

[0049] Thus, the present invention also relates to a pharmaceutical composition comprising a compound as described above and, optionally, a pharma- ceutical suitable carrier, vehicle, solvent or agent. The present invention also relates to a compound of the present invention for use in the prophylaxis, treatment or detection of a cancer characterized by TA-MUC1 expressing tumor cells. Preferably, the cancer treated or prevented by the compound of the present invention is selected from the group consisting of pancreatic cancer, prostate cancer, breast cancer, B-cell leukemia, multiple myeloma, colon cancer and ovarian cancer. As exemplified herein, a protein construct comprising binding sites for a first and a second polypeptide chain is capable of binding to mammalian TA-MUC1, preferably hu-TA-MUC1, and is suitable for the diagnosis and treatment of MUC1 expressing epithelial cancers.

[0050] The presence of a binding site within a compound is sufficient for the compound to recognize and bind to mammalian TA-MUC1.Thus, the invention includes any compound comprising the amino acid sequences of SEQ ID NO:1-SEQ ID NO:6, or homologues thereof from other species that are capable of binding to mammalian TA-MUC1, preferably hu-TA-MUC1.

[0051] The invention is further illustrated in the following examples.

[0052] Constructs The beneficial therapeutic and diagnostic effects of the compounds of the invention were tested using a protein construct carrying the binding site for hu-TA-MUC1 (=α-hu-TA-MUC1) and having the following composition: The first polypeptide sequence of the test compound has the amino acid sequence EVKLEESGGGLVQPGGSMKLSCVAS GFTFSDYW MNWVRQSPEKGLEWVAD IRLKSNNYAA HYAESVKGRFTVSRDDSKSSVYLQMNNLRTEDTGFYYC TFGNSFAY Contains WGQGTLVTVSA (sequence number 7).

[0053] The binding sites of the first polypeptide sequence corresponding to amino acid sequence SEQ ID NO:1 (GFTFSDYW), SEQ ID NO:2 (IRLKSNNYAA) and SEQ ID NO:3 (TFGNSFAY) are underlined.

[0054] The second polypeptide sequence of the test compound is QAVVTQESALTTSPGETVTLTCRSS TGAVTTNNY ANWVQEKPDHLFTGLIG GTN NRGPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHWV It contains the amino acid sequence of FGGGTKLTVL (SEQ ID NO:8).

[0055] The binding sites of the second polypeptide sequence corresponding to amino acid sequence SEQ ID NO:4 (TGAVTTNNY), ​​SEQ ID NO:5 (GTN) and SEQ ID NO:6 (ALWYSNHWV) are underlined.

[0056] The resulting compound is an scFv comprising the following amino acid sequence, which includes the binding sites of SEQ ID NO:1 to SEQ ID NO:6 (underlined): EVKLEESGGGLVQPGGSMKLSCVAS GFTFSDYW MNWVRQSPEKGLEWVAD IRLKSNNYAA HYAESVKGRFTVSRDDSKSSVYLQMNNLRTEDTGFYYC TFGNSFAY WGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSS TGAVTTNNY ANWVQEKPDHLFTGLIG GTN NRGPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHWV FGGGTKLTVL (sequence number 9).

[0057] The final leucine (L) of the amino acid sequence of SEQ ID NO: 9 can be followed by a histidine tag (HHHHHH) and / or a streptavidin II tag (WSHPQFEK) for purification and as a marker.

[0058] The binding sites in the first and second polypeptide sequences are connected by a linker amino acid. The amino acid sequence used to generate α-hu-TA-MUC1 is derived from the murine sequence.

[0059] result In Figure 1, the histological staining of human breast cancer biopsies for TA-MUC1 is shown. The mAb is under the construct (α-hu-TA-MUC1) mentioned above. 142 sections of human HR+ breast cancer tissue and 10 sections of human breast epithelial tissue were stained with α-hu-TA-MUC1. Treatment of healthy tissue with α-hu-TA-MUC1 was colorless in all cases. In contrast, all breast cancer tissue sections could be clearly stained with α-hu-TA-MUC1. A precise analysis of the staining of breast cancer tissue sections revealed that, depending on the intensity of the staining, two populations could be distinguished. Thus, a correlation was established between the strength of TA-MUC1 expression (staining intensity) and the survival of metastasis-free (MFS) and recurrence-free patients (RFS). The analysis of the cumulative survival of patients clearly correlated with the level of TA-MUC1 expression. High expression of the tumor marker was associated with a significantly worse survival rate. Immunohistological analysis confirmed the high specificity of the TA-MUC1-glycopeptide used as an antigen in the synthetic breast cancer vaccine. A preclinical breast cancer mouse model was established to evaluate the therapeutic efficacy of these vaccines in vivo.

[0060] PyMT transgenic (PyMT-tg) mice are a preclinical tumor model that is highly suitable for the development of therapeutic strategies to treat human breast cancer. The morphological similarities and the expression of biomarkers associated with poor outcome (overexpression of Her2 / neu, loss of estrogen and progesterone receptors) are consistent with those of humans. By crossing huMUC1-tg mice (C57BL / 6-TG(MUC1)79.24GEND / J) with PyMT-tg mice that express huMUC1 in all epithelial cells and develop aggressive palpable mammary tumors (breast tumors) at 14 weeks of age, we were able to develop a preclinical breast cancer model expressing hu-TA-MUC1, including 90% of all human epithelial breast cancers. This model offers the possibility of establishing active immunotherapy targeting hu-TA-MUC1 to treat breast cancer and provides the opportunity to apply the same or similar strategies in the clinical setting. The main obstacle for using modified self-antigens such as TA-MUC1 as vaccination targets is the autoimmune response. Logically, non-specific binding to healthy tissues by induced serum antibodies must be excluded. Therefore, with the aid of the same vaccine used in the following prophylactic vaccination study, the binding of the first generated mAb, α-hu-TA-MUC1, was confirmed using healthy mammary tissues of tumor-free huMUC1-tg mice by fluorescence microscopy. Binding to healthy mammary epithelial tissues, i.e., fully glycosylated huMUC1, and to breast tumor cells of PyMT-tg mice that do not express huMUC1, was not detectable. However, strong binding of α-hu-TA-MUC1 to breast tumor tissues of PyMTxhuMUC1-tg mice was observed, indicating that tumor-specific variants of huMUC1 were expressed from breast tumor cells of these mice (see Figure 2).

[0061] After demonstrating the expression of hu-TA-MUC1 in breast tumor cells of PyMTxhuMUC1-tg mice, primary tumor cell lines were established to develop a transplantable breast tumor model for preventive vaccination studies. As a negative control, primary tumor cell lines derived from PyMT tumor-bearing mice that did not express hu-TA-MUC1 were established in parallel. The expression of huMUC1 in primary tumor cells derived from PyMTxhuMUC1 and PyMT primary tumor cell lines was comparatively analyzed by qRT-PCR, and the surface expression of hu-TA-MUC1-glycoprotein was determined by FACS analysis using ohu-TA-MUC1.

[0062] Figure 3A shows the expression of huMUC1 mRNA in PyMTxhuMUC1 cells compared to that in human breast cancer cells (T47D) and human mammary epithelial cells (HMEC), the latter of which express fully glycosylated MUC. The strongest expression of huMUC1 mRNA was found in T47D cells, followed by PyMTxhuMUC1 cells. In the healthy breast epithelial cell line HMEC, there is much less expression of huMUC1 mRNA, indicating that huMUC1 is overexpressed in cancer cells. Tumor cells derived from PyMT-tg mice (PyMT cells) do not express huMUC1 mRNA, as do mouse B16F10 melanoma cells, which were selected as a negative control. The α-hu-TA-MUC1 mAb bound to significant amounts of PyMTxhuMUC1 breast tumor cells but failed to recognize PyMT breast tumor cells, indicating exclusive expression of the hu-TA-MUC1-glycoprotein in PyMTxhuMUC1 cells (see FIG. 3B).

[0063] In a next approach with the aid of a well-controlled preclinical spontaneous breast cancer model, the inventors first attempted to investigate whether preventive vaccination could lead to the recruitment of a sufficiently strong immune system to reject newly appearing breast tumors expressing hu-TA-MUC1. In this sense, PyMTxhuMUC1 and PyMT-tg mice were immunized three times with the synthetic vaccine at 2-week intervals.

[0064] Five days after the last immunization, blood samples were taken to determine antibody titers and isotypes. Tumor progression was monitored every 3 days. Tumor size was measured as length x width (mm2) due to their elliptical shape.

[0065] Figure 4A shows that immunized PyMTxhuMUC1-tg mice developed significantly smaller tumors than non-immunized PyMTxhuMUC1-tg mice. In contrast, immunized PyMT-tg mice did not show tumor reduction compared to untreated PyMT-tg mice.

[0066] Although all vaccinated mice showed similar hu-TA-MUC1-glycopeptide-specific antibody titers after the third vaccination (see Figure 4B), the induced humoral immune response showed significant tumor reduction efficiency only in PyMTxhuMUC1-tg mice. Analysis of isotype antibodies (see Figure 4C) confirmed that moderate IgG2b titers were generated.

[0067] Compared to immunized PyMT-tg mice, immunized PyMTxhuMUC1-tg mice showed a slight increase in IgM titers, possibly as a result of residual T cell tolerance in PyMTxhuMUC1 double-tg mice. Nevertheless, an overall strong humoral immune response was observed in the absence of an autoimmune response, confirming the results of previous vaccination studies in huMUC1-tg mice.

[0068] In additional experiments, we investigated the correlation between tumor reduction and survival time by preventive vaccination. For this purpose, PyMTxhuMUC1-tg mice were immunized three times starting at 6 weeks of age. Five days after the last immunization, blood samples were taken to determine IgG titers and antibody isotypes. Untreated PyMTxhuMUC1-tg mice were used as controls. Mice were sacrificed when the survival of the animals was severely restricted according to ethical guidelines.

[0069] Figure 5A shows that prophylactically vaccinated breast cancer-bearing mice survived approximately 13 days longer than untreated mice. ELISA analysis of total IgG titers (see Figure 5B) and isotype titers (see Figure 5C) showed induction of a strong and specific humoral immune response against hu-TA-MUC1, consistent with the results of previous experiments (see Figure 4). Serum from unimmunized tumor-bearing mice did not show any hu-TA-MUC1-glycopeptide antibody titers or binding to the MUC1-expressing T47D human breast cancer cell line or the PyMTxhuMUC1 primary tumor cell line (data not shown).

[0070] The presence of a binding site within the compound is sufficient for the compound to recognize and bind to mammalian TA-MUC1. Similar results were obtained with other compounds bearing the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:6, or homologues thereof from other species capable of binding to mammalian, preferably human, TA-MUC1.

[0071] conclusion The data show that α-hu-TA-MUC1 is a biologically and pharma- ceutical effective compound suitable for specifically detecting tumor tissue by binding to hu-TA-MUC1, as found in tumor cells but not in normal epithelial cells. Compounds carrying six binding sites of the invention allow selective detection of tumor tissue in tissue samples. The data also show that α-hu-TA-MUC1 is suitable for treating epithelial tumors, as shown by breast tumors. Prophylactic immunization prolongs the survival of patients suffering from breast tumors. Thus, the protein compounds of the invention are suitable for treating, preventing and / or diagnosing cancers expressing TA-MUC1, such as breast cancer, pancreatic cancer, prostate cancer, B-cell leukemia, multiple myeloma, colon cancer or ovarian cancer. Thus, any tumor cell expressing TA-MUC1 is a valid target for compounds of the invention carrying the six identified binding sites defined in SEQ ID NO: 1 to SEQ ID NO: 6. Our data show that high TA-MUC1 levels are inversely correlated with improved overall survival (MFS, RFS) in breast cancer patients.

[0072] Thus, the data clearly demonstrate that vaccine-based immunization strategies that induce the compounds of the invention have the potential to inhibit breast tumor progression and metastasis.

[0073] Materials and Methods Histological staining of human breast cancer biopsies A panel of 142 hormone receptor positive (HR+) breast cancer tissues from patients treated with tamoxifen in the adjuvant setting were examined for TA-MUC1 expression using α-hu-TA-MUC1 as a diagnostic tool. Samples were stained with α-hu-TA-MUC1 (1 pg / ml) and further stained with biotinylated goat anti-mouse antibody as a secondary antibody, followed by chromogenic reaction with SA-HPO. Human breast cancer cells (MCF-7), which strongly express TA-MUC1, served as a positive control, and paraffin sections of healthy glandular tissue and HR+ breast cancer tumors were examined. Furthermore, the magnitude of TA-MUC1 expression was scored in correlation with metastasis-free survival (MFS) and recurrence-free survival (RFS) according to the scoring system of Sinnet PR et al. (Sinnet PR et al., Comparative Study on The Diagnostic Accuracy of The Ripasa Score Over Alvarado Score In The Diagnosis of Acute Appendicitis Jebmh.2016;3(80):4318-21). Human MUC1-expressing preclinical breast cancer mouse model

[0074] C57BL / 6-TG(MUC1)79.24GEND / J mice (abbreviated as huMUC1-tg, The Jackson laboratory) express the human MUC1 gene on all epithelial cells and are crossed with mice of the breast cancer model line PyMT. Female mice positive for breast cancer antigens and the huMUC1 transgene (PyMTxhuMUC1-tg) develop several mammary tumors (4-5 tumors per mouse) expressing hu-TA-MUC1, comparable to human breast cancer stage IV after 14 weeks. Hu-TA-MUC1-glycopeptide-TTox vaccine

[0075] A synthetic MUC1-derived glycopeptide vaccine was generated to target specific MUC1 glycan patterns on human breast cancer cells, generating α-hu-TA-MUC1. The glycopeptide portion represents a 22-mer amino acid sequence from the VNTR region of huMUC1. It contains two immunodominant motifs, the APDTRP sequence and the STAPPA sequence. The serine at position 17 of the STAPPA motif was glycosylated with the STN carbohydrate antigen. The synthetic hu-TA-MUC1-glycopeptide was conjugated to the tetanus toxoid (TTox) carrier protein, which is known to induce strong helper T cell-specific immune responses in humans. The synthetic vaccine was dissolved in PBS and emulsified with incomplete Freund's adjuvant (IFA, Sigma Aldrich) (ratio 1:1). For one immunization, each mouse was intraperitoneally (ip) injected with 10 pg / 40 ml of emulsion.

[0076] Fluorescence microscopy - Binding of α-hu-TA-MUC1 to mouse breast tumor tissue ex vivo To establish the above PyMTxhuMUC1-tg mice as a preclinical breast cancer model for preventive vaccination studies, tumor tissues of PyMTxhuMUC1-tg mice were first tested for expression of hu-TA-MUC1-glycoprotein. For this purpose, the binding of fluorescently labeled α-hu-TA-MUC1 (AF647N: far-red fluorescent dye) was examined by fluorescence microscopy. Tumors were ex vivo removed, frozen in liquid nitrogen, and tissue sections were prepared. Sections were transferred to slides, stained with DAPI (1 / 1000) and AF647N-α-hu-TA-MUC1 (5 pg / ml), washed three times, and mounted on coverslips. Mammary gland sections of huMUC1-tg mice and tumor sections of PyMT-tg mice were used as controls. Fluorescent images were obtained using a confocal microscope LEICATCS SP8 equipped with a DMi8 microscope and a Zeiss LSM510META laser scanning microscope. Isolated ex vivo from PyMTxhuMUC1-tg and PyMT-tg mice

[0077] Generation of primary cell lines using cultured tumors With the aim of closely monitoring the growth of breast tumors after prophylactic vaccination, we generated primary tumor cell lines derived from tumors of PyMTxhuMUC1-tg mice. For this purpose, tumor tissues were extracted, digested with collagenase A (Roche, 2 mg / ml) and RQ1 DNAse (Promega, 1:2000) and cultured in IMDM+10% FCS+1% glutamine+1% sodium pyruvate. PyMTxhuMUC1 tumor cells and PyMT cells were harvested after 6 weeks. The expression of huMUC1 was examined by quantitative real-time PCR (qRT-PCR) and the binding of α-hu-TA-MUC1 to the hu-TA-MUC1-glycoprotein of both cell lines by FACS analysis (see below).

[0078] Determination of expression of huMUC1 by PyMTxhuMUC1 tumor cells by qRT-PCR and FACS analysis qRT-PCR: RT-2x10 6Total RNA from tumor cells was prepared using TRIzol Reagent (Invitrogen®, Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. RNA concentration and quality were measured photometrically (Eppendorf BioPhotometer plus). Reverse transcription was performed using MMLV reverse transcriptase (Thermo Scientific, Dreieich, Germany) according to the manufacturer's instructions, and qRT-PCR data were obtained using 5xHot Start Taq EVA Green (rock-free) mix (Axon, Kaiserslautern, Germany) in a MylQ iCycler (Biorad, Munich, Germany) with the provided software for data analysis (Bio-Rad iQ5 Standard Version 2.0) with the following primers: huMUCI: 5'-GTGCCCCCTAGCAGTACCG-3', rev: 5'-GACGTGCCCCTACAAGTTGG-3', and as reference gene: EF-1a: 5'-TGGATGCTCCAGGCCATAAGGA-3', rev: 5'-TGCTCTCGTGTTTGTCCTCCAG-3. The human breast cancer cell line T47D and human mammary epithelial cell line (HMEC) served as positive controls for huMUC1 expression. The B16F10 mouse melanoma cell line served as a negative control for huMUC1 expression.

[0079] FACS analysis: 2x105 PyMTxhuMUC1 and PyMT tumor cells were incubated with 1pg / ml α-hu-TA-MUC1, respectively, for 20 minutes at 4°C. The cells were washed twice with 100ml PBS and then incubated with the secondary antibody goat-α-mouse-lgG Alexa Fluor488 (diluted 1:1000 in PBS) and the fixable viability dye eFluor780 (diluted 1:1000 in PBS) for 20 minutes at 4°C to exclude false positive dead cells. Again, the cells were washed twice with 100ml PBS. The cells were then taken up in 100ml PBS and analyzed on a BD Biosciences FACS LSRII machine. For each sample, 10 4Cells were analyzed.

[0080] Vaccination of transgenic mice developing spontaneous breast tumors by administration of hu-TA-MUC1-glycopeptide-TTox vaccine PyMTxhuMUC1-tg and PyMT-tg mice (the latter control) were immunized three times at 2-week intervals (10 pg / 40 ml ip) starting at 6 weeks of age. Five days after each vaccination, blood was collected from the tail vein and serum was obtained. Antibody and isotype titers of induced antibodies were determined by ELISA (data from the first and second immunizations not shown). Immunized and untreated mice were sacrificed after 20 weeks and all tumors were isolated and quantified by calipers. Due to the oval shape of the tumors and their wavy surface, length x width was calculated (mm 2 ). Both PyMTxhuMUC1-tg and PyMT-tg mice developed 4-5 mammary tumors. Tumor size is shown as the average of the number of tumors in each mouse. Survival of immunized PyMTxhuMUC1-tg mice compared to untreated PyMTxhuMUC1-tg mice obtained several days after birth. Mice survival was monitored in accordance with ethical guidelines.

[0081] Analysis of hu-TA-MUC1-specific antibody titers and antibody isotypes by ELISA To analyze the IgG titers of hu-TA-MUC1-glycopeptide-specific induced antibodies, an ELISA was applied. 96-well plates (NuncMaxiSor® flat bottom) were incubated with 50 ml / well of hu-TA-MUC1-glycopeptide-BSA conjugate (2.5 pg / ml) / coating buffer (0.1 M Na2HP04 / water, pH 9.3) at 37° C. A washing step (3 times) was performed using 100 ml of blocking buffer (PBS with 1% BSA, 0.2% Tween-20). Subsequently, the coated plate was incubated for 20 min at 37° C. with 50 ml of blocking buffer to saturate the free binding sites. The antiserum was diluted in blocking buffer (1:100 in the first column of the 96-well plate, then serially diluted in a 1:1 ratio), pipetted into the ELISA plate and incubated for 45 min at 37° C. After three further washing steps, samples were incubated with biotinylated sheep-α-mouse IgG antibody (c=0.48 pg / ml, stock solution 250 pg / ml) in 50 ml / well of blocking buffer for 45 min at 37 °C. Plates were washed three times and incubated with 50 mL of blocking buffer containing streptavidin-horseradish peroxidase (1:1000) for 15 min. After three washes, each well was treated with citrate buffer (40 mM citric acid, 60 nM Na2HP04xH20, pH 4.5) containing 1 mg / ml 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 0.01% hydrogen peroxide (1:4000). The optical density of each well was measured at 410 nm using a spectrophotometer (Tecan Reader, Genios).

[0082] For isotype determination, hu-TA-MUC1-glycopeptide-specific antisera were analyzed after the third immunization and ELISA was performed using the protocol described above. The following secondary antibodies were used: biotinylated anti-mouse-lgM (eBioscience, clone eB121-15F9), biotinylated anti-mouse-lgG1 (BD Pharmingen, clone A85-1), biotinylated anti-mouse-lgG2b (BD Pharmingen, clone R19-15).

[0083] Antibody titers were calculated as half-maximum values ​​by non-linear regression (R2>0.97) followed by 4-parameter logistic curve analysis applying the software Graph Pad Prism6.

Claims

1. A compound comprising: - a first polypeptide sequence comprising at least three different amino acid sequences selected from the group consisting of GFTFSDYW (SEQ ID NO: 1), IRLKSNNYAA (SEQ ID NO: 2) and TFGNSFAY (SEQ ID NO: 3); a second polypeptide sequence comprising at least three different amino acid sequences selected from the group consisting of TGAVTTNNY (SEQ ID NO: 4), GTN (SEQ ID NO: 5) and ALWYSNHWV (SEQ ID NO: 6), The first polypeptide sequence comprises the amino acid sequence set forth in SEQ ID NO:7, and the second polypeptide sequence comprises the amino acid sequence set forth in SEQ ID NO:8; The compound is an scFv comprising the amino acid sequence set forth in SEQ ID NO:9, and is capable of binding to tumor-associated mucin-1 (TA-MUC1).

2. 2. The compound of claim 1, wherein the first polypeptide sequence is a portion of the variable region of the heavy chain (VH) of an antibody and the second polypeptide sequence is a portion of the variable region of the light chain (VL) of an antibody or antibody fragment.

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