ANTIBODIES BINDING TO Tn-MUC-1

Abstract

The present invention generally relates to antibodies that bind to Tn-MUC-1. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

Description

[0001] Case P38418

[0002] Antibodies binding to Tn-MUC-1

[0003] FIELD OF THE INVENTION

[0004] The present invention generally relates to antibodies that bind to Tn-MUC-1. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

[0005] BACKGROUND

[0006] MUC-1 (mucin 1, cell surface associated; CD227) is a high molecular weight transmembrane glycoprotein, expressed on the apical surfaces of most simple and glandular epithelia. Its extracellular domain contains a repeated stretch of highly glycosylated tandem repeats (TRs). A TR consists of a constant 20 amino acid sequence rich in serine (Ser) and threonine (Thr) residues, which are sites of attachment for O-linked glycans (O-glycans). Besides its expression on healthy cells, MUC-1 is also overexpressed by a variety of cancers, where it is distributed all over the cell surface, aberrantly glycosylated and shed in the tissue microenvironment.

[0007] The sugar chain structure of MUC-1 is different between normal and tumor cells. While MUC- 1 expressed by normal cells carries long, branched O-linked sugar chains, tumor cells express various simple and short sugar chain antigens due incomplete processing of O-glycans. O- glycosylation is initiated by N-acetylgalactosaminyltransferase, which utilizes UDP-GalNAc to add GalNAc to Ser / Thr residues in the protein backbone. In normal cells, the GalNAc residues attached to the protein backbone are further elongated by T synthase (Core 1 P-3- galactosyltransferase (C1GALT1)) to form the Core 1 structure (Gal-GalNAc-a-Ser / Thr). C1GALT1, requires the chaperone protein COSMC for folding and activity. In tumors, however, a typical MUC-1 glycostructure is the Tn antigen, which is formed by attachment via a-linkage of GalNAc to Ser / Thr (GalNAcal-O-Ser / Thr) and the increased expression of which has been attributed to inactive C1GALT1.

[0008] CL / 18.11.2025 Being one of the most prevalent aberrant glycoforms of MUC-1 found in cancer, the Tn glycoform of MUC-1 (Tn-MUC-1) represents an attractive target for cancer therapy, e.g. by “T cell bispecific antibodies” (also called “TCBs” herein).

[0009] The choice of target and the specificity of the targeting antibody is of utmost importance in cancer therapy to avoid on- and off-target toxicities.

[0010] Antibodies targeting Tn-MUC-1 are described in WO 2019 / 083506 (antibody “GO2”) and WO 2020 / 006449 (antibody “4AG”). There remains a need, however, for antibodies having favorable properties for therapeutic use in humans.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides novel antibodies that bind Tn-MUC-1 and have particularly favorable properties for production and therapeutic purposes. In particular, the antibodies can be well expressed, and also combine good produceability with good binding affinity to Tn- MUC-1 and good efficacy.

[0013] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody is a humanized antibody comprising a heavy chain variable region (VHMUC-I) and a light chain variable region (VLMUC-I) derived from a parental antibody, the parental antibody comprising the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 7, wherein the VLMUC-I comprises glycine at position 51 (Kabat numbering). In some aspects, the VLMUC-I comprises lysine at position 55 (Kabat numbering). In some aspects, the VHMUC-I comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 45, and the VLMUC-I comprises the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 53 and the LCDR 3 of SEQ ID NO: 56.

[0014] In a further aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable region (VHMUC-I) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 45, and a light chain variable region (VLMUC-I) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 53 and the LCDR 3 of SEQ ID NO: 56.

[0015] In some aspects, the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and a HCDR3 selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, a LCDR2 selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56.

[0016] In some aspects,

[0017] (i) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56;

[0018] (ii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0019] (iii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 41; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0020] (iv) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0021] (v) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 43; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0022] (vi) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56;

[0023] (vii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 44; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56; or

[0024] (viii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 50, and the LCDR3 of SEQ ID NO: 56.

[0025] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 65; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66.

[0026] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

[0027] In some aspects,

[0028] (i) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31;

[0029] (ii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;

[0030] (iii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;

[0031] (iv) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;

[0032] (v) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;

[0033] (vi) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31;

[0034] (vii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31; or (viii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC- i comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0035] In some aspects, the antibody is an IgG, particularly an IgGi, antibody. In some aspects, the antibody is a full-length antibody. In some aspects, the antibody is an antibody fragment selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab')2 molecule.

[0036] In some aspects, the antibody is a multispecific, particularly a bispecific, antibody, optionally wherein the multispecific antibody binds to Tn-MUC-1 and to CD3.

[0037] According to a further aspect of the invention there is provided an isolated polynucleotide encoding the antibody of the invention, and a host cell comprising the isolated polynucleotide of the invention.

[0038] In another aspect is provided a method of producing an antibody that binds to Tn-MUC-1, comprising culturing the host cell of the invention under conditions suitable for the expression of the antibody and optionally further comprising recovering the antibody from the host cell. The invention also encompasses an antibody that binds to Tn-MUC-1 produced by the method of the invention.

[0039] The invention further provides a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically acceptable carrier.

[0040] Also encompassed by the invention are methods of using the antibody and pharmaceutical composition of the invention. In one aspect, the invention provides an antibody or pharmaceutical composition according to the invention for use as a medicament. In one aspect is provided an antibody or pharmaceutical composition according to the invention for use in the treatment of cancer.

[0041] Also provided is the use of an antibody or pharmaceutical composition according to the invention in the manufacture of a medicament, and the use of an antibody or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of cancer. The invention also provides a method of treating cancer in an individual, comprising administering to said individual an effective amount of the antibody or pharmaceutical composition according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1. Schematic illustration of the bispecific antibody molecules prepared in the Examples. The tested bispecific antibody molecules were produced as “2+1 IgG CrossFab, inverted” (A) or “1+1 IgG CrossFab” (B) with charge modifications (VH / VL exchange in T-cell antigen binder, charge modifications in Tn-MUC-1 binders, EE = 147E, 213E; RK = 123R, 124K). Figure 2. RPAPG-S(GalNAc)-T(GalNAc)-APPA in complex with the Fab of antibody GO2. Paratope residues are shown in line representation, the glycosylated peptide is shown in ball- and-sticks representation. The first four residues of the peptide (RPAP) are not resolved in the electron density (no coordinates were assigned).

[0043] Figure 3. Binding of GO2-derived Tn-MUC-1 binders, following humanization, to target expressing T3M4 COSMC KO cells, as measured by flow cytometry (IgG format). Binding was compared with GO2 in TCB format (A) and IgG format (B). Antibodies bound to T3M4 COSMC KO cells were detected with a fluorescently labeled anti-human Fc specific secondary antibody.

[0044] Figure 4. Interaction of GO2-derived Tn-MUC-1 binders of the first round of humanization to Tn-glycosylated MUC-1 peptide Biotin-(O2Oc)2-RPAPG-S(GalNAc)-T(GalNAc)- APPAHGVT-amid as measured by SPR.

[0045] Figure 5. Binding of GO2-derived Tn-MUC-1 binders from the second round of humanization to MCF7 COSMC KO cells as measured by flow cytometry (IgG format). Binding was compared with the original GO2 in IgG format. Antibodies bound to MCF7 COSMC KO cells were detected with a fluorescently labeled anti-human Fc specific secondary antibody.

[0046] Figure 6. Interaction of GO2-derived Tn-MUC-1 binders of the third round of humanization to Tn-glycosylated MUC-1 peptide Biotin-(O2Oc)2-RPAPG-S(GalNAc)-T(GalNAc)- APPAHGVT-amid as measured by SPR.

[0047] Figure 7. Binding of GO2-derived Tn-MUC-1 TCBs to MCF7 COSMC KO cells as measured by flow cytometry. Binding was compared with the original GO2 and 4AG in TCB format with the “CD3orig” CD3 binder. Antibodies bound to MCF7 COSMC KO cells were detected with a fluorescently labeled anti-human Fc specific secondary antibody.

[0048] Figure 8. Target cell killing of MCF7 COSMC KO cells and T cell activation marker (CD25 upregulation) on CD4 T cells are shown in (A) and (B) respectively. PBMCs from a healthy donor were exposed to GO2-derived Tn-MUC-1 TCB antibodies and assessed after 72 hours of co-incubation with target cells. Tumor cell killing was measured by quantification of cell death using CytotoxGlo reagent (Promega) and CD25 upregulation was measured by flow cytometry.

[0049] Figure 9. Off-target activity was tested with HBEpiC from ScienCell Research Laboratories. PBMCs from a healthy donor were exposed to GO2-derived Tn-MUC-1 TCB antibodies and assessed after 72 hours of co-incubation with HBEpiC. Tumor cell killing was measured by quantification of cell death using CytotoxGlo reagent (Promega) (A) and CD25 upregulation was measured by flow cytometry (B).

[0050] Figure 10. Target cell killing of MCF7 COSMC KO cells and off-target activity on HBEpiC from ScienCell Research Laboratories are shown in (A) and (B), respectively. PBMCs from a healthy donor were exposed to GO2-derived Tn-MUC-1 TCB antibodies and assessed after 72 hours of co-incubation with the cells. Tumor cell killing was measured by quantification of cell death using CytotoxGlo reagent (Promega).

[0051] Figure 11. Cumulative cell death of MCF7 COSMC KO cells over 96 hours, in presence of PBMCs from a healthy donor and GO2-derived Tn-MUC-1 TCBs. Tumor cell killing was measured every 12 hours by quantification of cell death as described in the general methods section. Cumulative cell death was calculated as the sum of data points for all time points, per molecule and concentration.

[0052] Figure 12. The specificity of P1AH6390 (A) and P1AF2574 (B), as compared to GO2 (P1AA9754) (C), towards Tn-glycosylation was evaluated using HEK 293A COSMC KO + MUC1 cells and HEK 293 A + MUC1 cells. This was accomplished through the application of flow cytometry. Antibodies were detected with a fluorescently labeled anti-human Fc specific secondary antibody.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] I. DEFINITIONS

[0055] Terms are used herein as generally used in the art, unless otherwise defined in the following. “Tn-MUC-1” stands for the Tn-glycoform of MUC-1, particularly human MUC-1. MUC-1 (mucin 1, cell surface associated; CD227) is a high molecular- weight transmembrane glycoprotein. Human MUC-1 is described in UniProt (www.uniprot.org) accession no. Pl 5941 (entry version 263). The MUC-1 extracellular domain contains a repeated stretch of highly glycosylated tandem repeats (TRs). A TR consists of a constant 20 amino acid sequence rich in serine (Ser) and threonine (Thr) residues, which are sites of attachment for O-linked glycans (O-glycans). The attachment of GalNAc to Ser / Thr via a-linkage forms the Tn antigen (GalNAcal-O-Ser / Thr). In certain aspects, Tn-MUC-1 stands for a glycosylated 40-mer representing two copies of one of the tandem repeats present in MUC-1, glycosylated with GalNAc to form the Tn antigen (SEQ ID NO: 77):

[0056] HGV-T(GalNAc)-S(GalNAc)-APD-T(GalNAc)-RPAPG-S(GalNAc)-T(GalNAc)-APPA- HGV-T(GalNAc)-S(GalNAc)-APD-T(GalNAc)-RPAPG-S(GalNAc)-T(GalNAc)-APPA.

[0057] The terms “anti-Tn-MUC-1 antibody” and “an antibody that binds to Tn-MUC-1” refer to an antibody that is capable of binding Tn-MUC-1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting Tn-MUC-1. In some aspects, the extent of binding of an anti-Tn-MUC-1 antibody to an unrelated, non-Tn-MUC-1 protein is less than about 10% of the binding of the antibody to Tn-MUC-1 as measured, e.g., by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to Tn-MUC-1 has a dissociation constant (KD) of < 1 pM, < 500 nM, < 200 nM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM, particularly < 500 nM, more particularly < 200 nM.

[0058] By ’’specific binding“ is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. Suitable assays for determining the specificity of the antibody of the present invention are described herein, including in the Examples hereinbelow. In some aspects, the extent of binding of an antibody to an unrelated protein, including a different glycoform of MUC-1 (particularly non-glycosylated or core-1- glycosylated MUC-1), is less than about 10% of the binding of the antibody to the antigen as measured, e.g., by SPR.

[0059] The term ”antibody“ herein encompasses various antibody structures exhibiting the desired antigen-binding specificity, including but not limited to monoclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments.

[0060] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the antibodies forming this population are essentially identical, except for possible post-translational modifications arising e.g. during manufacturing and / or storage. These antibodies are directed against the same epitope (or the same group of epitopes in the case of multispecific monoclonal antibodies, e.g. the same pair of epitopes in the case of bispecific monoclonal antibodies). This definition expressly excludes polyclonal antibody preparations which are mixtures of antibodies directed against different epitopes. Monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to hybridoma methodology, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0061] The term “full-length antibody” refers to an antibody having the structure of an immunoglobulin comprising two light chains and two heavy chains, and comprising an Fc region as defined herein. In one aspect, the antibody is a full-length IgGi antibody.

[0062] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues. In certain aspects, a human antibody is derived from a non- human transgenic mammal, for example a mouse, a rat, or a rabbit. In certain aspects, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

[0063] A “humanized” antibody refers to an antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In one aspect, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody (the “parental” antibody), and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as “humanized variable region”. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived, i.e. the parental antibody), e.g., to restore or improve antibody specificity or affinity. The term “humanized” antibody also encompasses antibodies comprising certain mutations (e.g. amino acid substitutions) in the CDRs as compared to the CDRs of the parental antibody. In certain aspects, a humanized antibody comprises up to three (i.e. none, one, two or three) amino acid substitutions in each of its CDRs as compared to the parental antibody. In certain aspects, a humanized antibody comprises at least one CDR which is identical (i.e. does not comprises any amino acid substitutions as compared to the parental antibody) to the corresponding CDR of the parental antibody. In certain aspects, a humanized antibody comprises at least two CDRs (particularly one heavy chain CDR and one light chain CDR) which are identical to the corresponding CDRs of the parental antibody. A “humanized form” of an antibody, e.g. of a non-human antibody, refers to an antibody that has undergone humanization.

[0064] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (LC) and two identical heavy chains (HC) that are disulfide-bonded. From N- to C-terminus, each heavy chain has a heavy chain variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three heavy chain constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a light chain variable domain (VL), also called a variable light domain or a light chain variable region, followed by a light chain constant domain (CL).

[0065] An “antibody fragment” refers to a molecule other than a full-length antibody that comprises a portion of a full-length antibody that binds the antigen to which the full-length antibody binds. Examples of antibody fragments include but are not limited to Fv molecules, Fab molecules, Fab' molecules, Fab’-SH molecules, F(ab')2 molecules, diabodies, linear antibody molecules, single-chain antibody molecules (e.g., scFv and scFab molecules), and multispecific (e.g. bispecific) antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23 : 1126-1136 (2005).

[0066] A “multispecific antibody” is one having binding specificities for at least two different epitopes, i.e., different epitopes on different antigens or different epitopes on the same antigen. In one aspect, a multispecific antibody is a “bispecific antibody” having binding specificities for two epitopes. In one aspect, the multispecific antibody has three or more binding specificities. In one aspect, one of the binding specificities is for Tn-MUC-1 and the other specificity / ies is for any other antigen(s). In some aspects, the other specificity is for CD3. In one aspect, multispecific antibodies may bind to two (or more) different epitopes of Tn-MUC-1. Multispecific antibodies (e.g. bispecific antibodies) may comprise antibody fragments and / or one or more Fc region.

[0067] The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies binding to a particular epitope or determining an epitope bound by an antibody can be performed using methods known in the art such as, e.g., without limitation, alanine scanning, peptide blots (see, e.g., Reineke, Meth. Mol. Biol. 248: 443-463 (2004)), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see, e.g., Hochleitner et al. Prot. Sci. 9: 487-496 (2000)), cross-blocking (see, e.g., “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., Second Edition, NY (2014)), Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP) (US 2004 / 0101920), or x-ray crystallography.

[0068] An ’’isolated” antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography) methods. For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibody provided by the present invention is an isolated antibody.

[0069] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0070] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6thed., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-881 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0071] Glutamine or glutamate residues at the N-terminus of antibody heavy or light chains may be converted to pyro-glutamate spontaneously (see e.g. Liu et al., Journal of Pharmaceutical Sciences 97, 2426-2447 (2008), Rehder et al., Journal of Chromatography A 1102, 164-175 (2006), Chelius et al., Anal Chem 78, 2370-2376 (2006)). Hence, variable domains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N- terminus of an the antibody heavy or light chain, may comprise an N-terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Likewise, antibody heavy chains or light chains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus, may comprise an N-terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Accordingly, for each antibody heavy chain, light chain, or variable domain sequence disclosed herein that contains an N-terminal Q or E residue, the corresponding sequence with an N-terminal pyroE residue is also encompassed.

[0072] The term “complementarity determining region” or “CDR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity. Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). CDRs are defined by a variety of methods / systems by those skilled in the art. These systems and / or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and generally is the most commonly used. The Chothia definition is based on the location of the structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e.g., abYsis: http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) are available and known to those of skill in the art for analysis of antibody sequences and determination of CDRs.

[0073] Exemplary CDRs herein include (numbering of amino acid residues according to the reference cited, i.e. Chothia numbering for the Chothia and Contact definition, Kabat numbering for the Kabat definition and IMGT numbering for the IMGT definition):

[0074] (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3), according to Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) (“Chothia definition”);

[0075] (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35B (Hl), 50-65 (H2), and 95-102 (H3), according to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) NIH Publication 91-3242 (“Kabat definition”);

[0076] (c) antigen contacts occurring at amino acid residues 30-36 (LI), 46-55 (L2), 89-96 (L3), 30-35 (Hl), 47-58 (H2), and 93-101 (H3), according to MacCallum et al. J. Mol. Biol. 262: 732-745 (1996) (“Contact definition”); and

[0077] (d) CDRs occurring at amino acid residues residues 27-38 (LI), 56-65 (L2), 105-117 (L3), 27-38 (Hl), 56-65 (H2), and 105-117 (H3), according to Lefranc et al. Dev. Comp. Immunol. 27: 55-77 (2003) (“IMGT definition”).

[0078] Unless otherwise indicated, the CDRs are determined herein according to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) NIH Publication 91-3242 (hereinafter “Kabat 1991”). One of skill in the art will understand that the CDR designations can also be determined according to Chothia, MacCallum, Lefranc, or any other scientifically accepted definiti on / sy stem .

[0079] “Framework” or “FR” refers to variable domain residues other than complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following order in VH (or VL): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3- HCDR3(LCDR3)-FR4.

[0080] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat 1991.

[0081] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0082] A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat 1991.

[0083] The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively. Several of the antibody classes may be further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgG?, IgG , IgAi, and IgA?, with corresponding heavy chain constant domains yi (IgGi), y? (IgG?), 73 (IgGs), y4 (IgG ), ai (IgAi) and a? (IgA?). The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0084] The terms “constant region derived from human origin” or “human constant region” as used herein denotes a constant region of a human antibody, in particular a heavy chain constant region of a human antibody of the subclass IgGi, IgG?, IgG?, or IgG4 and / or a light chain kappa or lambda constant region. Such constant regions are well known in the state of the art and e.g. described in Kabat 1991. Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the numbering system as described in Kabat 1991. Specifically, the Kabat numbering system (referred to as “numbering according to Kabat” or “Kabat numbering” herein; see pages 647-660 of Kabat 1991) is used for the light chain constant domain of kappa and lambda isotype, and the Kabat EU index numbering system (referred to as “numbering according to Kabat EU index”, “Kabat EU numbering” or “Kabat EU index numbering” herein, see pages 661-723 of Kabat 1991) is used for the heavy chain constant domains.

[0085] The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case in particular where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU index numbering). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region are denoted herein without C-terminal lysine if not indicated otherwise. The corresponding sequence including a C-terminal lysine residue is also encompassed, however. Accordingly, in one aspect, a heavy chain including an Fc region as specified herein comprises an additional C-terminal lysine residue (K447, Kabat EU index numbering). Also encompassed is the corresponding sequence without the C-terminal glycine residue. Accordingly, in one aspect, a heavy chain including an Fc region as specified herein lacks the C-terminal glycine residue (G446, Kabat EU index numbering). In such a heavy chain, the C-terminal amino acid residue may be proline (P445, Kabat EU index numbering) or proline amide (P445-NH2, Kabat EU index numbering). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system, also called the EU index, as described in Kabat 1991.

[0086] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), down regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0087] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is Surface Plasmon Resonance (SPR).

[0088] A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0089] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to one or more cytotoxic agent(s).The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, nonconservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3- methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.

[0090] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU5 10087 and is described in WO 2001 / 007611.

[0091] Unless otherwise indicated, for purposes herein, percent (%) amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448), W. R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227- 258), and Pearson et. al. (Genomics 46 (1997) 24-36) and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down. shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global proteimprotein) program and default options (BLOSUM50; open: -10; ext: - 2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0092] The term “polynucleotide” or “nucleic acid molecule” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2101823 Bl).

[0093] An “isolated” polynucleotide (or nucleic acid) refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated polynucleotide includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0094] “Isolated polynucleotide (or nucleic acid) encoding an antibody” refers to one or more polynucleotide molecules encoding antibody heavy and light chains (or fragments thereof), including such polynucleotide molecule(s) in a single vector or separate vectors, and such polynucleotide molecule(s) present at one or more locations in a host cell.

[0095] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0096] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Suitable host cells may include, for example, CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NSO cells, lymphocytic cells, prokaryotic cells such as E. coli, and other eukaryotic hosts such as plant cells and fungi. Human host cells are included with the proviso that they are not used within the human body. In one aspect, the host cell of the invention is a eukaryotic cell, particularly a mammalian cell. In one aspect, the host cell is not a cell within a human body.

[0097] The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Specifically, the term refers to a preparation of the antibody of the invention and one or more pharmaceutically acceptable carriers or excipients.

[0098] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, surfactant and / or preservative.

[0099] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0100] An “individual” or “subject” is a mammal. In one aspect, the individual or subject is a human. In one aspect, the individual is in need of treatment with the medicament or antibody disclosed herein.

[0101] An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired treatment as defined above.

[0102] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0103] II. COMPOSITIONS AND METHODS

[0104] The invention provides antibodies that bind Tn-MUC-1. The antibodies show good produceability, combined with other favorable properties for therapeutic application, e.g. with respect to affinity and efficacy. Antibodies of the invention as useful, e.g., for the treatment of diseases such as cancer. A. Anti-Tn-MUC-1 antibodies

[0105] In one aspect, the invention provides an antibody that binds to Tn-MUC-1. In one aspect, provided is an isolated antibody that binds to Tn-MUC-1. In one aspect, the invention provides an antibody that specifically binds to Tn-MUC-1. In one aspect, the antibody is a monoclonal antibody that binds to Tn-MUC-1. In one aspect, the antibody is a humanized antibody that binds to Tn-MUC-1.

[0106] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody is a humanized antibody comprising a heavy chain variable region (VHMUC-I) and a light chain variable region (VLMUC-I) derived from a parental antibody, the parental antibody comprising the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 7, wherein the VLMUC-I comprises glycine at position 51 (Kabat numbering).

[0107] In certain aspects, the antibody shows increased expression yield as compared to the parental antibody. In some aspects, the antibody shows at least 2-fold, at least 3-fold, at least 5-fold or at least 10-fold, particularly about 5-fold increased expression yield as compared to the parental antibody. In some aspects, the antibody shows an expression yield of at least 10 mg / L cell culture. In specific aspects, such expression yield is after Protein A affinity chromatography, and / or in HEK293 suspension cell culture.

[0108] In certain aspects, the antibody comprises up to three (i.e. none, one, two or three) amino acid substitutions in each of its complementarity determining regions (CDRs) as compared to the parental antibody. In certain aspects, the antibody comprises at least one CDR which is identical (i.e. does not comprise any amino acid substitutions as compared to the parental antibody) to the corresponding CDR of the parental antibody. In certain aspects, the antibody comprises at least two CDRs (particularly one heavy chain CDR and one light chain CDR) which are identical to the corresponding CDRs of the parental antibody. In some aspects, the parental antibody comprises the heavy chain CDR (HCDR) 1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 37, the HCDR3 of SEQ ID NO: 39, the light chain CDR (LCDR) 1 of SEQ ID NO: 47, the LCDR2 of SEQ ID NO: 49 and the LCDR3 of SEQ ID NO: 56.

[0109] In some apects, the VLMUC-I comprises lysine at position 55 (Kabat numbering).

[0110] In some aspects, the antibody binds to Tn-MUC-1 with at least essentially equal affinity as the parental antibody. In some aspects, the antibody binds to Tn-MUC-1 with a KD of less than 500 nM, particularly less than 200 nM, as measured by surface plasmon resonance at 25°C. In some aspects, the VHMUC-I comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 45, and the VLMUC-I comprises the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 53 and the LCDR 3 of SEQ ID NO: 56.

[0111] In a further aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable region (VHMUC-I) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 45, and a light chain variable region (VLMUC-I) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 53 and the LCDR 3 of SEQ ID NO: 56.

[0112] In some aspects of the antibody according to the invention, the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and a HCDR3 selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, a LCDR2 selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56.

[0113] In some aspects,

[0114] (i) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56;

[0115] (ii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0116] (iii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 41; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0117] (iv) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;

[0118] (v) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 43; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56; (vi) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56;

[0119] (vii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 44; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56; or

[0120] (viii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 50, and the LCDR3 of SEQ ID NO: 56.

[0121] In particular aspects, the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56.

[0122] In further particular aspects, the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56.

[0123] In some aspects, the antibody is a humanized antibody. In some aspects, the VHMUC-I and / or the VLMUC-I is a humanized variable region. In some aspects, the VHMUC-I and / or the VLMUC-I comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

[0124] In some aspects, the VHMUC-I comprises one or more heavy chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 57, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 57, a FR2 comprising the amino acid sequence of SEQ ID NO: 58, a FR3 comprising the amino acid sequence of SEQ ID NO: 59, and a FR4 comprising the amino acid sequence of SEQ ID NO: 60.

[0125] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 65. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 65.

[0126] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, particularly the amino acid sequence of SEQ ID NO: 27. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, particularly the amino acid sequence of SEQ ID NO: 27. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, particularly the amino acid sequence of SEQ ID NO: 27. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn-MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0127] In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 65. Optionally, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 65, including post-translational modifications of that sequence.

[0128] In some aspects, the VHMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, particularly the amino acid sequence of SEQ ID NO: 27. Optionally, the VHMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, particularly the amino acid sequence of SEQ ID NO: 27, including post-translational modifications of that sequence.

[0129] In some aspects, the VLMUC-I comprises one or more light chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of a light chain variable region sequence selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 61, a FR2 comprising the amino acid sequence of SEQ ID NO: 62, a FR3 comprising the amino acid sequence of SEQ ID NO: 63, and a FR4 comprising the amino acid sequence of SEQ ID NO: 64.

[0130] In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 66. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 66.

[0131] In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31, particularly the amino acid sequence of SEQ ID NO: 31. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31, particularly the amino acid sequence of SEQ ID NO: 31. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31, particularly the amino acid sequence of SEQ ID NO: 31. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn- MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 16, SEQ ID NO: 20 or SEQ ID NO: 31. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0132] In some aspects, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 66. Optionally, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 66, including post-translational modifications of that sequence.

[0133] In some aspects, the VLMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31, particularly the amino acid sequence of SEQ ID NO: 31. Optionally, the VLMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31, particularly the amino acid sequence of SEQ ID NO: 31, including post-translational modifications of that sequence.

[0134] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 65; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 66. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 65; and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 66.

[0135] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31. In some aspects, the VHMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; and / or the VLMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

[0136] In some aspects,

[0137] (i) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31;

[0138] (ii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0139] (iii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0140] (iv) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0141] (v) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0142] (vi) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31;

[0143] (vii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31; or

[0144] (viii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16.

[0145] In some aspects,

[0146] (i) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31; (ii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0147] (iii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 26 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0148] (iv) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0149] (v) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 28 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0150] (vi) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31;

[0151] (vii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 29 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31; or

[0152] (viii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 16.

[0153] In particular aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27 and the VLMUC- i comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. In further particular aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31.

[0154] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and the VLMUC- i comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20. In further aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20.

[0155] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 65, and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 66. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 65 and / or the VLMUC- i comprises the amino acid sequence of SEQ ID NO: 66. In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31. In some aspects, the VHMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and / or the VLMUC-I comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

[0156] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising

[0157] (i) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31;

[0158] (ii) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0159] (iii) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0160] (iv) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; (v) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0161] (vi) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31;

[0162] (vii) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31; or

[0163] (viii) a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16.

[0164] In some aspects,

[0165] (i) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31;

[0166] (ii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0167] (iii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 26 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0168] (iv) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0169] (v) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 28 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20;

[0170] (vi) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31; (vii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 29 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31; or

[0171] (viii) the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 16.

[0172] In a particular aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27 and a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. In particular aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 31.

[0173] In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13 and a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 20.

[0174] In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 65, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 66.

[0175] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of a VH selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of a VL selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

[0176] In a further aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising (i) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 31; (ii) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 20;

[0177] (iii) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 26, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 20;

[0178] (iv) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 20;

[0179] (v) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 28, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 20;

[0180] (vi) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 31;

[0181] (vi) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 29, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 31; or

[0182] (viii) a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 16.

[0183] In a particular aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 31.

[0184] In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 20.

[0185] In some aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 65, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 66. In some aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of a VH selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of a VL selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

[0186] In some aspects,

[0187] (i) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 27, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 31;

[0188] (ii) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 13, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 20;

[0189] (iii) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 26, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 20;

[0190] (iv) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 27, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 20;

[0191] (v) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 28, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 20;

[0192] (vi) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 13, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 31;

[0193] (vii) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 29, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 31; or

[0194] (viii) the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 13, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 16.

[0195] In particular aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 27, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 31. In other aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 13, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 20.

[0196] In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 65, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 65, and / or the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 66, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 66.

[0197] In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of a VH selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of a VH selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and / or the VLMUC-I comprises the light chain CDR sequences of a VL selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of a VL selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

[0198] In further aspects,

[0199] (i) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 27, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 31, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 31;

[0200] (ii) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 13, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 20, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 20; (iii) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 26, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 26, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 20, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 20;

[0201] (iv) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 27, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 20, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 20;

[0202] (v) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 28, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 28, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 20, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 20;

[0203] (vi) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 13, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 31, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 31;

[0204] (vii) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 29, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 29, and / or the VLMUC- i comprises the light chain CDR sequences of the VL of SEQ ID NO: 31, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 31; or

[0205] (viii) the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 13, and / or the V MUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 16, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 16.

[0206] In particular aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VH of SEQ ID NO: 27. In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a framework of at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO: 27. In other aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a framework of at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO: 27.

[0207] In further particular aspects, the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 31 and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VL of SEQ ID NO: 31. In some aspects, the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 31 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 31. In other aspects, the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 31 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 31.

[0208] In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VH of SEQ ID NO: 13. In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO: 13. In other aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO: 13.

[0209] In further aspects, the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 20 and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VL of SEQ ID NO: 20. In some aspects, the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 20 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 20. In other aspects, the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 20 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 20.

[0210] Additional anti-Tn-MUC-1 antibodies

[0211] In a different aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable region (VHMUC-I) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 42, and a light chain variable region (VLMUC-I) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 49 and the LCDR 3 of SEQ ID NO: 56.

[0212] In some aspects, the antibody is a humanized antibody. In some aspects, the VHMUC-I and / or the VLMUC-I is a humanized variable region.

[0213] In some aspects, the VHMUC-I and / or the VLMUC-I comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

[0214] In some aspects, the VHMUC-I comprises one or more heavy chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of the heavy chain variable region sequence of SEQ ID NO: 27. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 57, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 57, a FR2 comprising the amino acid sequence of SEQ ID NO: 58, a FR3 comprising the amino acid sequence of SEQ ID NO: 59, and a FR4 comprising the amino acid sequence of SEQ ID NO: 60.

[0215] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 27. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 27. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 27. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn- MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 27. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0216] In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27. Optionally, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27, including post-translational modifications of that sequence.

[0217] In some aspects, the VLMUC-I comprises one or more light chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of the light chain variable region sequence of SEQ ID NO: 32. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 61, a FR2 comprising the amino acid sequence of SEQ ID NO: 62, a FR3 comprising the amino acid sequence of SEQ ID NO: 63, and a FR4 comprising the amino acid sequence of SEQ ID NO: 64.

[0218] In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 32. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 32. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 32. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn- MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 32. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0219] In some aspects, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 32. Optionally, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 32, including post-translational modifications of that sequence.

[0220] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27; and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 32.

[0221] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27, and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC- i comprises the amino acid sequence of SEQ ID NO: 32.

[0222] In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 32.

[0223] In some aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 27, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 32.

[0224] In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 27, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 27, and / or the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 32, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 32.

[0225] In a further different aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable region (VHMUC-I) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 40, and a light chain variable region (VLMUC-I) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 55 and the LCDR 3 of SEQ ID NO: 56.

[0226] In some aspects, the antibody is a humanized antibody. In some aspects, the VHMUC-I and / or the VLMUC-I is a humanized variable region.

[0227] In some aspects, the VHMUC-I and / or the VLMUC-I comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

[0228] In some aspects, the VHMUC-I comprises one or more heavy chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of the heavy chain variable region sequence of SEQ ID NO: 13. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 57, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 57, a FR2 comprising the amino acid sequence of SEQ ID NO: 58, a FR3 comprising the amino acid sequence of SEQ ID NO: 59, and a FR4 comprising the amino acid sequence of SEQ ID NO: 60.

[0229] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 13. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 13. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn- MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 13. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ IDNO: 13. Optionally, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13, including post-translational modifications of that sequence.

[0230] In some aspects, the VLMUC-I comprises one or more light chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of the light chain variable region sequence of SEQ ID NO: 33. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 61, a FR2 comprising the amino acid sequence of SEQ ID NO: 62, a FR3 comprising the amino acid sequence of SEQ ID NO: 63, and a FR4 comprising the amino acid sequence of SEQ ID NO: 64.

[0231] In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 33. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 33. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn- MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 33. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0232] In some aspects, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 33. Optionally, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 33, including post-translational modifications of that sequence.

[0233] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13; and / or the V MUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13; and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 33.

[0234] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13, and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC- i comprises the amino acid sequence of SEQ ID NO: 33.

[0235] In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 33.

[0236] In some aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 13, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 33.

[0237] In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 13, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 13, and / or the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 33, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 33.

[0238] In still a further different aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable region (VHMUC-I) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 54, and the HCDR 3 of SEQ ID NO: 40, and a light chain variable region (VLMUC-I) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 49 and the LCDR 3 of SEQ ID NO: 56.

[0239] In some aspects, the antibody is a humanized antibody. In some aspects, the VHMUC-I and / or the VLMUC-I is a humanized variable region. In some aspects, the VHMUC-I and / or the VLMUC-I comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

[0240] In some aspects, the VHMUC-I comprises one or more heavy chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of the heavy chain variable region sequence of SEQ ID NO: 6. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 57, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some aspects, the VHMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 57, a FR2 comprising the amino acid sequence of SEQ ID NO: 58, a FR3 comprising the amino acid sequence of SEQ ID NO: 59, and a FR4 comprising the amino acid sequence of SEQ ID NO: 60.

[0241] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 6. In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 6. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn-MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 6. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0242] In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 6. Optionally, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 6, including post-translational modifications of that sequence.

[0243] In some aspects, the VLMUC-I comprises one or more light chain framework sequence (i.e. the FR1, FR2, FR3 and / or FR4 sequence) of the light chain variable region sequence of SEQ ID NO: 9. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61, a FR2 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62, a FR3 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, and / or a FR4 comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some aspects, the VLMUC-I comprises a framework region (FR) 1 comprising the amino acid sequence of SEQ ID NO: 61, a FR2 comprising the amino acid sequence of SEQ ID NO: 62, a FR3 comprising the amino acid sequence of SEQ ID NO: 63, and a FR4 comprising the amino acid sequence of SEQ ID NO: 64.

[0244] In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9. In some aspects, the VLMUC-I comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 9. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to Tn-MUC-1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 9. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0245] In some aspects, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 9. Optionally, the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 9, including post-translational modifications of that sequence.

[0246] In some aspects, the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 6; and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 9.

[0247] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, and / or a light chain variable region (VLMUC-I) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9. In some aspects, the VHMUC-I comprises the amino acid sequence of SEQ ID NO: 6 and / or the VLMUC-I comprises the amino acid sequence of SEQ ID NO: 9.

[0248] In another aspect, the invention provides an antibody that binds to Tn-MUC-1, comprising a heavy chain variable region (VHMUC-I) comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 6, and a light chain variable region (VLMUC-I) comprising the light chain CDR sequences of the VL of SEQ ID NO: 9.

[0249] In some aspects, the VHMUC-I comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 6, and the VLMUC-I comprises the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 9.

[0250] In some aspects, the VHMUC-I comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 6, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VH of SEQ ID NO: 6, and / or the VLMUC-I comprises the light chain CDR sequences of the VL of SEQ ID NO: 9, and a framework of at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98%, sequence identity to the framework sequence of the VL of SEQ ID NO: 9.

[0251] In some aspects, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a VHMUC-I sequence as in any of the aspects provided above, and a VLMUC- i sequence as in any of the aspects provided above.

[0252] In some aspects, the antibody comprises a human constant region. In some aspects, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG class immunoglobulin molecule comprising a human CHI, CH2, CH3 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 74 and 75 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 76 (human IgGl heavy chain constant domains CH1-CH2-CH3).

[0253] In one aspect, the anti-Tn-MUC-1 antibody comprises an Fc region. In one aspect, the Fc region is an IgG Fc region, more particularly an IgGi Fc region. In another aspect, the Fc region is an IgG4Fc region. In one aspect, the Fc region is an IgG4Fc region comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In one aspect, the Fc region is a human Fc region. In a further aspect, the Fc region is a human IgGi Fc region. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 73. In one aspect, additionally the C-terminal lysine (Lys447) is present. In another aspect, the C- terminal glycine (Gly446) is absent. In such aspect, the C-terminal amino acid residue may be proline (Pro445) or proline amide (Pro445-NH2).

[0254] In one aspect, the anti-Tn-MUC-1 antibody is a full-length antibody, e.g., a full-length IgGi antibody or other antibody class or isotype as defined herein. In one aspect, the antibody is an IgG, particularly an IgGi, antibody. In another aspect, the antibody is an antibody fragment selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab’)2 molecule; particularly a Fab molecule or a scFv molecule. In one aspect, the antibody fragment is a diabody, a triabody or a tetrabody.

[0255] In a further aspect, an anti-Tn-MUC-1 antibody according to any of the above aspects may incorporate any of the features, singly or in combination, as described below.

[0256] / . Antibody Affinity

[0257] In one aspect, an antibody provided herein has a dissociation constant (KD) of < IpM, < 500 nM, < 250 nM, < 200 nM, < 100 nM, < 50 nM, < 25 nM, < 20 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10'6M or less, e.g., from 10'6M to 10'9M, e.g., from 10'7M to 10'8M), particularly a KD of < 500 nM, more particularly a KD of < 200 nM.

[0258] The binding (affinity) of the antibody to a target antigen or an Fc receptor can be determined for example by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (Cytiva), and receptors or target antigens such as may be obtained by recombinant expression. Alternatively, binding of antibodies to different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS). Exemplary assays for determining antigen binding affinity by SPR, as well as target cell binding activity by FACS are described in Example 1 herein. A specific illustrative and exemplary aspect for measuring binding activity to Tn-MUC- 1 is described in the following.

[0259] In particular aspects, the binding affinity (KD) of an antibody of the invention to Tn-MUC-1 is determined by SPR at 25°C. For example, SPR may be performed as follows: SPR is performed on a Biacore 8K instrument (Cytiva) at 25°C with HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20, Cytiva, Freiburg / Germany) as running buffer.

[0260] Biotinylated Tn-MUC-1 peptide (Tn-glycosylated MUC-1 peptide Biotin-(O2Oc)2-RPAPG- S(GalNAc)-T(GalNAc)-APPAHGVT-amid, SEQ ID NO: 79) is directly coupled to streptavidin linked to single strain DNA on a CAP chip by using the biotin CAPture kit (Cytiva, Freiburg / Germany). In a first step, on all eight channels single strain DNA, coupled to streptavidin, is hybridized to the complementary single strain DNA on a CAP chip (contact time: 300 s, flow: 2 pl / min). Subsequently, biotinylated Tn-MUC-1 peptides are captured via streptavidin biotin coupling on flow channels 1 up to 8 (contact time: 180 s, flow: 10 pl / min) with a concentration of 5 nM each. The antibody of the invention, in the form of a Fab fragment, is passed over the surface with an increasing concentration series from 0 - 50 - 100 - 200 - 400 and 500 nM with a flow of 30 pl / min over all eight flow channels. Association and dissociation are monitored for 180 s and 600 s respectively. The chip surface is regenerated after every cycle by using one injection of the corresponding regeneration mix (8 M guanidine chloride and 0.25 M sodium hydroxide) for 120 s. Bulk refractive index differences are corrected by subtracting the response obtained on the reference flow cell 1 of each channel, which contains the Tn-MUC-1 peptide, but no Tn-MUC-1 antibody captured on it. Data fitting and KD determination is performed using Biacore Insight Software (Cytiva).

[0261] 2. Antibody Fragments

[0262] In one aspect, the anti-Tn-MUC-1 antibody provided herein is or comprises an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, or F(ab’)2 fragment.

[0263] In one aspect, the antibody fragment is a Fab fragment containing each the heavy and light chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI).

[0264] In one aspect, the antibody fragment is a scFv fragment containing each the heavy and light chain variable domains (VH and VL, respectively), connected by a linker.

[0265] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of a full-length antibody as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein. 3. Humanized Antibodies

[0266] In one aspect, the anti-Tn-MUC-1 antibody provided herein is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In one aspect, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0267] 4. Library-Derived Antibodies

[0268] In one aspect, the anti-Tn-MUC-1 antibody provided herein is derived from a library. Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8: 1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8: 1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)) and in Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ (2003)).

[0269] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as singlechain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993). Furthermore, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US Patent Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0270] Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. in Methods in Molecular Biology 503: 135-56 (2012) and in Cherf et al. in Methods in Molecular Biology 1319: 155-175 (2015) as well as in Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942 (1997).

[0271] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0272] 5. Multispecific Antibodies

[0273] In one aspect, the anti-Tn-MUC-1 antibody provided herein is a multispecific, e.g. a bispecific, antibody.

[0274] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., Carter et al., J Immunol Meth 248, 7-15 (2001)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011 / 034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98 / 50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444- 6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

[0275] Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. The bispecific antibody may also include a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to Tn-MUC-1 as well as another different antigen, or two different epitopes of Tn-MUC-1 (see, e.g., US 2008 / 0069820 and WO 2015 / 095539). Bispecific antibodies also include a “DutaFab” wherein a single pair of a VH domain and a VL domain may bind to two different epitopes and wherein one paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 and the other paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 (see, e.g., WO 2012 / 163520).

[0276] Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH / VL domains (see e.g., WO 2009 / 080252 and WO 2015 / 150447), the CH1 / CL domains (see e.g., WO 2009 / 080253) or the complete Fab arms (see e.g., WO 2009 / 080251, WO 2016 / 016299). Also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010- 20. In one aspect, the multispecific antibody comprises a cross-Fab fragment. The term “cross- Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab (i.e. heavy and light chain) pairing. Exemplary Fab pairing amino acid mutations are Q39E (Kabat numbering) and S183K (Kabat EU numbering) in the heavy chain and Q38K (Kabat numbering) and V133E (Kabat EU numbering) in the light chain, or Q39K (Kabat numbering) and S183E (Kabat EU numbering) in the heavy chain and Q38E (Kabat numbering) and V133K (Kabat EU numbering) in the light chain (see e.g. WO 2016 / 172485). Further Fab pairing mutations include 124K, 124R or 124H (Kabat numbering) and 123K, 123R or 123H (Kabat numbering) in the light chain and 147E or 147D (Kabat EU numbering) and 213E or 213D (Kabat EU numbering) in the heavy chain (see e.g. WO 2015 / 150447).

[0277] To promote the correct association of heavy chains in asymmetric multispecific (e.g. bi specific) antibodies, their heavy chains may be engineered to comprise e.g. sterically (“knob-in-hole”) or electrostatically complementary amino acid mutations, salt bridges, and / or disulfide bonds. The knob-in-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996), Atwell et al., J. Mol. Biol. 270, 26 (1997), Merchant et al., Nat Biotechnol 16, 677-681 (1998), and Carter, J Immunol Meth 248, 7-15 (2001). In one aspect, a multispecific (e.g. bispecific) antibody comprising a human IgGi Fc domain may comprise the“knob” mutation T366W on the first heavy chain, and “hole” mutations Y407V and optionally T366S and L368A (all Kabat EU numbering) on the second heavy chain (T366W / T366S:L368A:Y407V). Additionally, the antibody may comprise a S354C substitution on the first heavy chain and a Y349C substitution (both Kabat EU numbering) on the second heavy chain, forming a disulfide bond ((T366W:S354C / Y349C:T366S:L368A:Y407V).

[0278] Further examples of amino acid mutations (e.g. substitutions) that may be comprised in multispecific (e.g. bispecific) antibodies include the substitution S228P (Kabat EU numbering) in antibodies comprising an IgG4 Fc region, e.g. for preventing Fab arm exchange (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0279] Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67: 95-106 (2015)).

[0280] A particular type of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activating, invariant component of the T cell receptor (TCR) complex, such as CD3, for retargeting of T cells to kill target cells. Hence, in one aspect, the antibody provided herein is a multispecific antibody, particularly a bispecific antibody, wherein one of the binding specificities is for Tn-MUC-1 and the other is for the component of the TCR complex, e.g. CD3. Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, the so-called “BiTE” (bispecific T cell engager) molecules wherein two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinity retargeting) molecules which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are full-length hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Particular T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) el203498.

[0281] 6. Antibody Variants

[0282] In one aspect, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a. Substitution, Insertion, and Deletion Variants

[0283] In one aspect, antibody variants having one or more amino acid substitutions are provided. Substitutions are possible in the CDRs, FRs, or constant regions. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0284] Amino acids may be grouped according to common side-chain properties:

[0285] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0286] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0287] (3) acidic: Asp, Glu;

[0288] (4) basic: His, Lys, Arg;

[0289] (5) residues that influence chain orientation: Gly, Pro;

[0290] (6) aromatic: Trp, Tyr, Phe.

[0291] Conservative substitutions will entail exchanging members of one of these classes for another member in the class.

[0292] One type of substitutional variant involves substituting one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0293] Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots”, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In one aspect of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4- 6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0294] In one aspect, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0295] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionine residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody. b. Glycosylation variants

[0296] In one aspect, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0297] Where the antibody comprises an Fc region, the oligosaccharide attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Jennewein et al. Trends in Immunology 38:P358-372 (2017). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In one aspect, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

[0298] In one aspect, antibody variants are provided having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as “afucosylated” oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have enhanced FcyRIIIa receptor binding and / or improved effector function, in particular enhanced ADCC function. See, e.g., US 2003 / 0157108; US 2004 / 0093621.

[0299] In one aspect, the antibody having a non-fucosylated oligosaccharide is produced by a FUT8KO CHO cell line. See, e.g., Jennewein et al. Trends in Immunology 38:P358-372 (2017).

[0300] In a further aspect, antibody variants are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0301] Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764. c. Fc region variants

[0302] In one aspect, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGi, IgG?, IgGs or IgG4 Fc region sequence) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0303] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase binding to human FcyR (e.g. FcyRIIIa) and / or effector function (e.g. ADCC), e.g., substitutions at positions 239, 298, 330, 332, 333 and / or 334 of the Fc region (Kabat EU numbering of residues). In one aspect, the substitutions are S298A, E333A and K334A in an Fc region derived from a human IgGi Fc region (see e.g. Shields et al. (2001) J. Biol. Chem. 276, 6591-6604). In one aspect, the substitutions are S239D, I332E and optionally A330L in an Fc region derived from a human IgGi Fc region (see e.g Lazar et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 4005-4010).

[0304] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which reduce binding to human FcyR (e.g. FcyRIIIa) and / or effector function (e.g. ADCC), e.g., substitutions at positions 228, 233, 234, 235, 265, 267, 297, 329 and / or 331 of the Fc region (Kabat EU numbering of residues).

[0305] In one aspect, the substitutions are L234A and L235A (LALA) in an Fc region derived from a human IgGi Fc region. In one aspect, the Fc region further comprises a D265A and / or P329G substitution. In one aspect, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgGi Fc region. See, e.g., WO 2012 / 130831, Schlothauer et al., Protein Eng Des Sei 29, 457-466 (2016). In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgGi Fc region.

[0306] In one aspect, the substitutions are S228P and L235E (SPLE) in an Fc region derived from a human IgG4 Fc region. In one aspect, the Fc region further comprises a P329G substitution. In one aspect, the substitutions are S228P, L235E and P329G (SPLE-PG) in an Fc region derived from a human IgG4 Fc region. See, e.g., WO 2012 / 130831, Schlothauer et al., Protein Eng Des Sei 29, 457-466 (2016).

[0307] In one aspect, the substitution is N297A (NA), N297G (NG) or N297Q (NQ) in an Fc region derived from a human IgGi Fc region. In one aspect, the Fc region further comprises a D265A substitution. In one aspect, the substitutions are D265A and N297A (DANA), or D265A and N297G (DANG) in an Fc region derived from a human IgGi Fc region.

[0308] In one aspect, the substitutions are E233P, L234V, L235A and G236del in an Fc region derived from a human IgGi Fc region (see e.g. Armour et al., Eur. J. Immunol. 29, 2613-2624 (1999)). In one aspect, the Fc region further comprises a N297G or S267 substitution.

[0309] In one aspect, the substitutions are L234F, L235E and D265A (FEA), or L234F, L235E and P331 S (FES), in an Fc region derived from a human IgGi Fc region.

[0310] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which decrease binding to human FcRn and / or serum half-life of the antibody, e.g. substitutions at positions 253, 310 and / or 435 (Kabat EU numbering of residues). In one aspect, the substitutions are 1253 A, H310A and H435A (AAA) in an Fc region derived from a human IgGi Fc region.

[0311] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase binding to human FcRn and / or serum half-life of the antibody, e.g. substitutions at positions 252, 254, 256, 428 and / or 434 (Kabat EU numbering of residues). In one aspect, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgGi Fc region (see, e.g., Dall’Acqua et al. J Biol Chem 281, 23514-23524 (2006); WO 2002 / 60919). In one aspect, the substitutions are M428L and N434S (see e.g. Zalevsky et al. NatBiotech 28, 157-159 (2010); WO 2009 / 086320). In one aspect, the substitutions are M428L and N434A.

[0312] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which reduce binding to rheumatoid factor, e.g. substitutions at positions 424, 436, 438 and / or 440 (Kabat EU numbering of residues). In one aspect, the substitutions are Q438R and S440E (RE) (see e.g. Maeda et al. MABS 9, 844-853 (2017)).

[0313] In one aspect, amino acid substitution(s) which increase FcRn binding are combined with amino acid substitution(s) which reduce binding to rheumatoid factor, as described e.g. in Maeda et al. MABS 9, 844-853 (2017). In one aspect, the substitutions are M428L, N434A and Y436T, or M428L, N434S and Y436T, in an Fc region derived from a human IgGi Fc region. In one aspect, the substitutions are N434A, Q438R, S440E, and optionally Y436T or Y436V, in an Fc region derived from a human IgGi Fc region. In one aspect, the substitutions are M428L, N434A, Q438R, S440E, and optionally Y436T or Y436V, in an Fc region derived from a human IgGi Fc region.

[0314] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase the antibody’s isoelectric point (pl), e.g. substitutions at positions 311 and / or 434 (Kabat EU numbering of residues). In one aspect, the substitutions are Q311R and P343R.

[0315] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase affinity to human FcyRIIb, e.g. substitutions at positions 234, 235, 236, 238, 250, 264, 268, 295, 307, 326 and / or 330 (Kabat EU numbering of residues). In one aspect, the substitutions are L235W, G236N, H268D, Q295L, K326T and A330K, or L234Y, P238D, T250V, V264I, T307P and A330K.

[0316] For additional Fc region mutations see e.g. Abdeldaim and Schindowski, Pharmaceutics 15(10): 2402 (2023).

[0317] The C-terminus of the Fc region of an antibody provided herein may be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the Fc region may also be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one aspect, the C-terminus of the Fc region is a shortened C-terminus ending with the amino acid residue P. In one aspect, the C-terminus of the Fc region is a shortened C- terminus ending with the amino acid residues PG. In one aspect, an antibody comprising an Fc region as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, Kabat EU numbering of amino acid positions). In one aspect, an antibody comprising an Fc region as specified herein, comprises a C-terminal glycine residue (G446, Kabat EU numbering of amino acid positions). d. Antibody Derivatives

[0318] In one aspect, an antibody provided herein may be further modified to contain additional non- proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Nonlimiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)poly ethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0319] 7. I minu noconjugates

[0320] The invention also provides immunoconjugates comprising an anti-Tn-MUC-1 antibody as described herein conjugated (chemically bonded) to one or more therapeutic agents (payloads) such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), immune stimulants or radioactive isotopes.

[0321] In one aspect, an immunoconjugate is an antibody-drug conjugate (ADC) in which an anti-Tn- MUC-1 antibody is conjugated to one or more of the therapeutic agents mentioned above. The antibody is typically connected to one or more of the therapeutic agents (payloads) using linkers. The number of payloads conjugated to the antibody is defined by the average drug-to-antibody ratio (DAR).

[0322] In one aspect, an immunoconjugate comprises an anti-Tn-MUC-1 antibody as described herein conjugated to a cytotoxic agent selected from microtubule inhibitors or DNA damaging agents. Examples for microtubule inhibitors include the auristatins, e.g. monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), and maytansinoid derivatives, e.g. DM1 (emtansine) and DM4 (soravtansine). Examples for DNA damaging agents include calicheamicin derivatives, e.g. N-acetyl gamma calicheamicin, pyrrolobenzodiazepines, e.g. SG3249 (tesirine), and topoisomerase I inhibitors such as, e.g. SN-38 (govitecan), deruxtecan (Dxd), or other camptothecin (CPT) derivatives.

[0323] In another aspect, an immunoconjugate comprises an anti-Tn-MUC-1 antibody as described herein conjugated to an immune stimulant. Examples for immune stimulants are TLR7 / 8 agonists, TLR7 agonists or STING agonists. In another aspect, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.

[0324] In one aspect, an immunoconjugate comprising an anti-Tn-MUC-1 antibody as described herein comprises one or more linkers. A linker may comprise one or more linker components. Exemplary linker components include 4-(A-maleimidom ethyl) cyclohexane- 1 -carboxylate (“MCC”), N-succinimidyl-4(2-pyridylthio)-pentanoate (“SPP”), 6- maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), and amino acid units such as valine-citrulline (“val-cif ’ or “vc”), alanine-phenylalanine (“ala-phe”), valine-alanine (“val-ala”) and glycine-glycine- phenylalanine -glycine (“GGFG”). Linker components also include spacer components such as p-aminobenzylcarbamate (“PABC”) or polyethylene glycol moieties (PEGs) comprising subunits of the formula (-CEE-CEE-O-jn, wherein n is an integer from 2 to 20.

[0325] The linker may be a “non-cleavable linker”, meaning that intracellular proteolytic degradation of the immunoconjugate will be required to release the therapeutic agent, or it may be a “cleavable linker” facilitating release of the payload in the cell. For example, an immunoconjugate comprising an antibody as described herein may comprise one or more cleavable linkers that may include chemically labile linkers (e.g. disulfides), acid-cleavable linkers (e.g. hydrazone linkers), and enzyme-cleavable linkers (e.g. protease-sensitive linkers) that are cleaved by lysosomal enzymes such as cathepsins (e.g. cathepsin B) that are present in the target cells.

[0326] Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional coupling agents such as active esters (e.g. A-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), A-succinimidyl-3-(2-pyridyldithio) butanoate (SPDB), succinimidyl-4-(7V- maleimidom ethyl) cyclohexane- 1 -carboxylate (SMCC), and A-succinimidyl 6- maleimidohexanoate), aldehydes (e.g. 4-(4-acetyl-phenoxy) butanoic acid (AcBut)) and azido derivatives (e.g. 6-Azidohexylamine).

[0327] Conjugation is typically achieved either via antibody cysteine (thiol) or lysine (amino) residue side chains. While lysine side chains are often unmodified, the thiol groups of cysteine residues are almost exclusively found as disulphide bonds and therefore require selective reduction before conjugation can occur.

[0328] In one aspect, it may be desirable to create cysteine engineered antibodies, e.g., THIOMAB™ antibodies, in which one or more residues of an antibody are substituted with cysteine residues. In a particular aspect, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described herein. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016 / 040856. Examples for THIOMAB™ mutations for antibody drug conjugates (ADCs) include an additional cysteine in the light chain (LC) at position KI 49 according to Kabat numbering (LC K149C mutation), or additional cysteines in the light chain at position 149 according to Kabat numbering and in the heavy chain (HC) at positions 174 and 373 according to Kabat EU numbering (LC K149C; HC L174C and Y373C mutations).

[0329] B. Recombinant Methods and Compositions

[0330] Antibodies may be produced using recombinant methods and compositions. For these methods one or more isolated polynucleotide(s) encoding the anti-Tn-MUC-1 antibody are provided.

[0331] In one aspect, two polynucleotides are prepared, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such polynucleotide(s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chain(s) of the antibody). These polynucleotides may be on the same expression vector or on different expression vectors.

[0332] In case of a bispecific antibody with heterodimeric heavy chains four polynucleotides are prepared, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc region polypeptide. The four polynucleotides may be comprised in one or more nucleic acid molecules or expression vectors. Such polynucleotide(s) encode an amino acid sequence comprising the first VL and / or an amino acid sequence comprising the first VH including the first heteromonomeric Fc region and / or an amino acid sequence comprising the second VL and / or an amino acid sequence comprising the second VH including the second heteromonomeric Fc region of the antibody (e.g., the first and / or second light and / or the first and / or second heavy chains of the antibody). These polynucleotides can be on the same expression vector or on different expression vectors, normally these polynucleotides are located on two or three expression vectors, i.e. one vector can comprise more than one of these polynucleotides. In one aspect, isolated polynucleotides encoding an antibody as used in the methods as described herein are provided. In one aspect, a method of making an anti-Tn-MUC-1 antibody is provided, wherein the method comprises culturing a host cell comprising polynucleotide(s) encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (including the host cell culture medium).

[0333] For recombinant production of an anti-Tn-MUC-1 antibody, polynucleotides encoding the antibody, e.g., as described above, are prepared and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotides may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) or produced by recombinant methods or obtained by chemical synthesis.

[0334] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ, pp. 245-254 (2003), describing expression of antibody fragments in E. coli. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0335] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, T.U., Nat. Biotech. 22 1409-1414 (2004) and Li, H. et al., Nat. Biotech. 24: 210-215 (2006).

[0336] Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0337] Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing technology for producing antibodies in transgenic plants).

[0338] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ, pp. 255-268 (2004).

[0339] In one aspect, the host cell is a eukaryotic cell, e.g., a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) cell.

[0340] In one aspect, the host cell is an isolated host cell. In one aspect, the host cell is not a cell within a human body.

[0341] When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, in one aspect, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, affinity chromatography (e.g. protein A chromatography), size exclusion chromatography, anion or cation exchange chromatography, mixed-mode chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and lectin chromatography.

[0342] C. Assays

[0343] Antibodies provided herein may be identified, screened for, or characterized for their physical / chemi cal properties and / or biological activities by various assays known in the art.

[0344] 1. Binding assays

[0345] The binding (affinity) of the antibody to a target antigen or an Fc receptor can be determined for example by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (Cytiva), and receptors or target antigens such as may be obtained by recombinant expression. Alternatively, binding of antibodies to different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS). Exemplary assays for determining antigen binding affinity by SPR, as well as target cell binding activity by FACS are described in Example 1 herein. A specific illustrative and exemplary aspect for measuring binding activity to Tn-MUC- 1 is described in the following.

[0346] In some aspects, the binding affinity of an antibody of the invention to Tn-MUC-1 is determined by SPR as follows:

[0347] SPR is performed on a Biacore 8K instrument (Cytiva) at 25°C with HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20, Cytiva, Freiburg / Germany) as running buffer.

[0348] Biotinylated Tn-MUC-1 peptide (Tn-glycosylated MUC-1 peptide Biotin-(O2Oc)2-RPAPG- S(GalNAc)-T(GalNAc)-APPAHGVT-amid, SEQ ID NO: 79) is directly coupled to streptavidin linked to single strain DNA on a CAP chip by using the biotin CAPture kit (Cytiva, Freiburg / Germany). In a first step, on all eight channels single strain DNA, coupled to streptavidin, is hybridized to the complementary single strain DNA on a CAP chip (contact time: 300 s, flow: 2 pl / min). Subsequently, biotinylated Tn-MUC-1 peptides are captured via streptavidin biotin coupling on flow channels 1 up to 8 (contact time: 180 s, flow: 10 pl / min) with a concentration of 5 nM each. The antibody of the invention, in the form of a Fab fragment, is passed over the surface with an increasing concentration series from 0 - 50 - 100 - 200 - 400 and 500 nM with a flow of 30 pl / min over all eight flow channels. Association and dissociation are monitored for 180 s and 600 s respectively. The chip surface is regenerated after every cycle by using one injection of the corresponding regeneration mix (8 M guanidine chloride and 0.25 M sodium hydroxide) for 120 s. Bulk refractive index differences are corrected by subtracting the response obtained on the reference flow cell 1 of each channel, which contains the Tn-MUC-1 peptide, but no Tn-MUC-1 antibody captured on it. Data fitting and KD determination is performed using Biacore Insight Software (Cytiva).

[0349] 2. Activity assays

[0350] Biological activity of the (multi specific) antibodies of the invention can be measured by various assays as described in the Examples. Biological activities may for example include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the induction of lysis of target cells such as tumor cells (by T cells), and the induction of tumor regression and / or the improvement of survival. Exemplary assays for determining T cell activation as well as T cell mediated tumor cell killing are described in Example I herein. D. Methods and Compositions for Diagnostics and Detection

[0351] In one aspect, any of the anti-Tn-MUC-1 antibodies provided herein is useful for detecting the presence of Tn-MUC-1 in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In one aspect, a biological sample comprises a cell or tissue, such as a cancer tissue.

[0352] In one aspect, an anti-Tn-MUC-1 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of Tn-MUC-1 in a biological sample is provided. In one aspect, the method comprises contacting the biological sample with an anti-Tn-MUC-1 antibody as described herein under conditions permissive for binding of the anti-Tn-MUC-1 antibody to Tn-MUC-1, and detecting whether a complex is formed between the anti-Tn-MUC-1 antibody and Tn-MUC-1. Such a method may be an in vitro or in vivo method. In one aspect, an anti-Tn-MUC-1 antibody is used to select subjects eligible for therapy with an anti-Tn-MUC-1 antibody, e.g., where Tn-MUC-1 is a biomarker for selection of patients.

[0353] Exemplary disorders that may be diagnosed using an antibody of the invention include cancer. In one aspect, labeled anti-Tn-MUC-1 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.

[0354] E. Pharmaceutical Compositions

[0355] In a further aspect, provided are pharmaceutical compositions comprising any of the anti-Tn- MUC-1 antibodies provided herein, e.g., for use in any of the below therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the anti-Tn-MUC-1 antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the anti-Tn-MUC-1 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0356] Pharmaceutical compositions (formulations) of an anti-Tn-MUC-1 antibody as described herein can be prepared by combining the antibody with pharmaceutically acceptable carriers or excipients known to the skilled person. See, for example Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) and Falconer R.J., Biotechnology Advances 37: 107412 (2019). Exemplary pharmaceutical compositions of an anti-Tn-MUC-1 antibody as described herein are lyophilized, aqueous, frozen, etc.

[0357] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0358] The pharmaceutical composition herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0359] The pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0360] F. Therapeutic Methods and Routes of Administration

[0361] Any of the anti-Tn-MUC-1 antibodies provided herein may be used in therapeutic methods.

[0362] In one aspect, an anti-Tn-MUC-1 antibody for use as a medicament is provided. In a further aspect, an anti-Tn-MUC-1 antibody for use in treating cancer is provided. In one aspect, an anti-Tn-MUC-1 antibody for use in a method of treatment is provided. In one aspect, the invention provides an anti-Tn-MUC-1 antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the anti-Tn- MUC-1 antibody. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In a further aspect, the invention provides for the use of an anti-Tn-MUC-1 antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for treatment of cancer. In a further aspect, the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0363] In a further aspect, the invention provides a medicament (adapted) for the treatment of cancer, comprising an anti-Tn-MUC-1 antibody. In one aspect, the medicament is (adapted) for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0364] In a further aspect, the invention provides a method for treating cancer. In one aspect, the method comprises administering to an individual having cancer an effective amount of an anti- Tn-MUC-1 antibody. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0365] An “individual” according to any of the above aspects is preferably a human. The individual according to any of the above aspects may be in need of the medicament and / or treatment with the anti-Tn-MUC-1 antibody. According to any of the above aspects, the cancer may be Tn- MUC-1 -expressing cancer, and / or a cancer selected from the group consisting of ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, breast cancer and lung cancer.

[0366] In a further aspect, the invention provides pharmaceutical compositions comprising any of the anti-Tn-MUC-1 antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the anti-Tn-MUC-1 antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the anti-Tn-MUC-1 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0367] Antibodies of the invention can be administered alone or used in a combination therapy. For instance, the combination therapy includes administering an antibody of the invention and administering at least one additional therapeutic agent (e.g. one, two, three, four, five, or six additional therapeutic agents). In one aspect, the combination therapy comprises administering an antibody of the invention and administering at least one additional therapeutic agent, such as a further anti-cancer agent.

[0368] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate pharmaceutical composition(s)), and separate administration, in which case administration of the antibody of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent or agents. In one aspect, administration of the anti-Tn-MUC-1 antibody and administration of an additional therapeutic agent occur within about one, two, three, four, five, or six days, within about one, two or three weeks, or within about one month, of each other. In one aspect, the antibody and additional therapeutic agent are administered to the patient on Day 1 of the treatment. Antibodies of the invention can also be used in combination with radiation therapy.

[0369] An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, intranasal and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0370] Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or from about 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0371] For the treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. The progress of this therapy is easily monitored by conventional techniques and assays.

[0372] G. Articles of Manufacture

[0373] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. III. SEQUENCES

[0374] IV. EXAMPLES

[0375] The following are examples of methods and compositions of the invention. It is understood that various other aspects may be practiced, given the general description provided above.

[0376] Example 1 - General methods

[0377] Recombinant DNA Techniques

[0378] Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New

[0379] York, 1989. The molecular biological reagents were used according to the manufacturer’s instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242. Gene Synthesis

[0380] Desired gene segments were synthesized at Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow subcloning into the respective expression vectors. All constructs for secretory proteins were designed with a 5 ’-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells.

[0381] Production of IgG and TCB antibodies

[0382] The DNA sequences encoding the variable heavy and light chain regions of the Tn-MUC-1 binders (and, where applicable, the CD3 binders) were cloned into mammalian expression vectors using conventional cloning techniques.

[0383] CD3 bispecific antibodies are also referred to herein as “T cell bispecific antibodies”, “TCB antibodies”, or “TCBs”. A schematic illustration of the CD3 bispecific antibodies prepared in these examples is given in Figure 1A.

[0384] The antibodies described herein were produced using shaking flasks with FedBatch mode. The recombinant production was performed by transient transfection of Expi293™ Cells in a defined, serum-free medium. For transfection, ExpiFectamine™ 293 Transfection Kit was used (Gibco). Cell culture supernatants were harvested 7-12 days after transfection.

[0385] Quantification of protein titer

[0386] The protein titer of supernatant samples was determined by affinity chromatography using a POROS A 20 pm column, 2.1 x 30 mm (Life Technologies, Carlsbad, CA, USA) on a High Performance Liquid Chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, MA, USA). The supernatant was loaded onto the column equilibrated with 0.2 M Na2HPO4, pH 7.4, followed by elution with 0.1 M citric acid, 0.2 M NaCl, pH 2.5. Titers were quantified by measuring absorption at 280 nm, and subsequently calculating the protein concentration by comparing the elution peak area (under the curve) of the analyte with a reference standard curve.

[0387] Purification oflgG and TCB antibodies Proteins were purified from cell culture supernatants referring to standard protocols. In brief, Fc containing proteins were purified from cell culture supernatants by Protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5 or PBS; elution buffer: 20 mM, 25 mM or 50 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0 followed by immediate pH neutralization of the sample. The protein was concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated protein was separated from monomeric protein by size exclusion chromatography (SEC) in 20 mM histidine, 140 mM sodium chloride, pH 6.0. If needed, cation-exchange chromatography (cIEX) was applied as an additional purification step after Protein-A affinity chromatography to achieve the essential purity.

[0388] Analytics oflgG and TCB antibodies

[0389] The concentrations of purified proteins were determined by measuring the absorption at 280 nm using the mass extinction coefficient calculated on the basis of the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. Purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of a reducing agent using a LabChipGXII or LabChip GX Touch (Perkin Elmer). Determination of the aggregate content was performed by HPLC chromatography at 25°C using analytical size-exclusion column (TSKgel G3000 SW XL or UP-SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH2PO4, 250 mM KC1 pH 6.2, 0.02% NaN3).

[0390] The IgGs and TCB antibodies were purified by Protein A and size exclusion chromatography. The final quality was good for all molecules with varying monomer content from around 70% to almost 100% monomer content and >90% purity on CE-SDS. In conclusion, all IgGs and TCB antibodies were produced in good quality.

[0391] Cell engineering

[0392] Human cell lines expressing the O-linked Tn-glycan were generated by inactivating the C1GALT1C1 gene, also known as COSMC, which codes for the Cl GALT 1 -specific chaperone 1. The inactivation was accomplished by applying the CRISPR / Cas9 KO technology either by co-transfecting plasmid DNA encoding for Cas9 and a CIGALTICl-specific gRNA or by transfecting a CIGALTICl-specific Cas9 / gRNA ribonucleoprotein. Successful gene disruption was confirmed by sequencing the PCR-amplified target DNA and Tn glycan-specific flow cytometry. The CRISPR / Cas9 mediated knock-out of C1GALT1C1 was performed in the pancreatic cancer cell line T3M4, the breast cancer cell line MCF-7 and the human embryonic kidney cell line 293 A. The resulting cell lines expressing the Tn glycan were named T3M4 COSMC KO, MCF7 COSMC KO and HEK 293A COSMC KO, respectively. Since 293A cells express MUC-1 only at very low levels, two additional cell lines were created by overexpressing human MUC-1 fused to an intracellular GFP domain in the COSMC KO as well as wt background. The resulting cell lines were named HEK 293 A COSMC KO + MUC1 and HEK 293 A + MUC 1.

[0393] Surface plasmon resonance

[0394] Binding of humanized anti-Tn-MUC-1 immunoglobulins to biotinylated Tn-MUC-1 peptides was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25 °C with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20; Cytiva, Freiburg / Germany).

[0395] Biotinylated Tn-MUC-1 peptides were directly coupled to streptavidin linked to single strain DNA on a CAP chip by using the biotin CAPture kit (Cytiva, Freiburg / Germany). In a first step, on all four channels single strain DNA, coupled to streptavidin, was hybridized to the complementary single strain DNA on a CAP chip (contact time: 150 s, flow: 2 pl / min). Subsequently, biotinylated Tn-MUC-1 peptides were captured via streptavidin biotin coupling on flow channels 2, 3, and 4 (contact time: 200 s, flow: 5 pl / min) with a concentration of 200 nM each. The humanized anti-Tn-MUC-1 immunoglobulins passed the surface at a concentration of 300 nM with a flow of 30 pl / min over all four flow channels. Association and dissociation were monitored for 180 s and 800 s, respectively. The chip surface was regenerated after every cycle by using one injection of the corresponding regeneration mix (6 M guanidine chloride and 0.25 M sodium hydroxide) for 120 s. Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell 1.

[0396] The following protocol was applied to assess the interaction of monovalent antibody fragments (Fabs) and biotinylated Tn-MUC-1 peptides in a quantitative manner allowing for the determination of kinetic constants: These SPR experiments were performed on a Biacore 8K (Cytiva) at 25°C with HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20, Cytiva, Freiburg / Germany) as running buffer.

[0397] Biotinylated Tn-MUC-1 peptides were directly coupled to streptavidin linked to single strain DNA on a CAP chip by using the biotin CAPture kit (Cytiva, Freiburg / Germany). In a first step, on all eight channels single strain DNA, coupled to streptavidin, was hybridized to the complementary single strain DNA on a CAP chip (contact time: 300 s, flow: 2 pl / min). -n-

[0398] Subsequently, biotinylated Tn-MUC-1 peptides were captured via streptavidin biotin coupling on flow channels 1 up to 8 (contact time: 180 s, flow: 10 pl / min) with a concentration of 5 nM each. Respective anti-Tn-MUC-1 binders passed the surface with an increasing concentration series from 0 - 50 - 100 - 200 - 400 and 500 nM with a flow of 30 pl / min over all eight flow channels. Association and dissociation were monitored for 180 s and 600 s respectively. The chip surface was regenerated after every cycle by using one injection of the corresponding regeneration mix (8 M guanidine chloride and 0.25 M sodium hydroxide) for 120 s. Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell 1 of each channel. Data fitting and KD determination was performed using Biacore Insight Software (Cytiva).

[0399] Target cell binding

[0400] Binding of molecules on target expressing cell lines was measured by flow cytometry.

[0401] Indirect labeling requires two incubation steps, firstly with a primary antibody, then with a secondary detection antibody. The secondary antibody is conjugated to a fluorescent dye.

[0402] Target cells were harvested, washed once with FACS buffer and re-suspended at a density of 2 x 106cells / ml. 100 pl of the cell suspension was transferred to a 96 well round bottom plate. Molecules were diluted in FACS buffer at indicated concentrations and before 50 pl of molecules were added to the target cells, plates were centrifuged and supernatant was discarded. The plates were incubated for 30 minutes at 4°C with the primary antibody.

[0403] The plates were centrifuged for 3 min at 600 x g, supernatant was removed and cells were washed with PBS. This was repeated 2 times before 50 pl of the secondary antibody mix containing Zombie Aqua™ Fixable Viability Kit (Biolegend, #423102) and PE anti-human Fc (Jackson #109-116-170) was added. After 20 minutes incubation at room temperature plates were washed twice with PBS and cells were finally resuspended in 100 pl FACS buffer per well. The cells were acquired using a BD FACSymphony A3 flow cytometer. EC50 values have been calculated by using a logistic regression curve fit with TIBCO Spotfire Roche pRED 12.0.4.

[0404] Primary T cell activation and T cell mediated tumor cell killing

[0405] The functional characterization of T cell bispecific antibodies (TCBs) was performed by assessing T cell activation and killing of tumor cells.

[0406] Human peripheral blood mononuclear cells (PBMCs) were isolated out of buffy coats obtained from the ‘Blutspende Zentrum Zurich’. The buffy coats from healthy donors were provided in 50 ml bags and PBMCs were isolated with a density gradient media (Lymphoprep™, Stemcell #07801) by following manufacturer's protocol. PBMCs were resuspended at a density of 6 x 106cells / ml in assay medium (RPMI 1640, Gibco #72400) containing 10% FBS. 50 pl of the cell suspension was transferred into a 96 well round bottom plate. Target cells were detached using trypsin (Gibco), washed once with PBS and re-suspended at a density of 0.3 x 106cells / ml in assay medium. 100 pl of the cell suspension was transferred to the plate containing the PBMCs.

[0407] Antibodies were diluted in assay medium at indicated concentrations and molecules were added to the wells. Assay medium was added to appropriate wells to reach equal volumes in each well. The plates were incubating for 48 h at 37°C, 5% CO2.

[0408] Dead cells release proteases and their accumulation correlates with the killing capacity of T cells. T cell killing was assessed by the detection of dead cell protease accumulation with the CytoTox-Glo™ Cytotoxicity Assay (Promega, #G9291).

[0409] After the incubation of the cells, CytoTox-Glo™ substrate was adjusted to room temperature before measurement. 75 pl of supernatant per well was transferred to a 96 well white flat bottom plate for analysis. 25 pl of the substrate was subsequently added to each well and after 15 minutes incubation at room temperature, luminescence was measured using a Perkin Elmer EnVision® 2104 instrument.

[0410] T cell activation induced upregulation of CD25 and CD69 expression was detected by flow cytometry. The plates were centrifuged for 3 min at 600 x g, supernatant was removed and cells were washed with PBS. This was repeated twice before 50 pl of the antibody mix containing Zombie Aqua™ Fixable Viability Kit (Biolegend, #423102), anti-huCD4 PerCP / Cy5.5 (Biolegend, #344608), anti-huCD8a BV711 (Biolegend, #301044), anti-huCD25 PE (Biolegend, #302606) and anti-huCD69 FITC (Biolegend, #310904) was added to each well. The plates were incubated for 30 min at 4°C. Afterwards, the cells were washed twice with FACS buffer and re-suspended in 150 pl of FACS buffer per well. The cells were acquired using a BD FACSymphony A3 flow cytometer.

[0411] Example 2 - Humanization of murine anti-Tn-MUC-1 antibody GO2

[0412] The murine anti-Tn-MUC-1 antibody “GO2” (see WO 2019 / 083506, incorporated herein by reference in its entirety) was humanized. Suitable human acceptor frameworks were identified by querying a BLASTp database of human V- and J-region sequences for the murine input sequences of antibody GO2 (cropped to the variable part). Selective criteria for the choice of human acceptor framework were sequence homology, same or similar CDR lengths, and the estimated frequency of the human germline, but also the conservation of certain amino acids at the VH-VL domain interface. Following the germline identification step, the CDRs of the murine input sequences were grafted onto the human acceptor framework regions. Each amino acid difference between these initial CDR grafts and the parental antibodies was rated for possible impact on the structural integrity of the respective variable region, and “back mutations” towards the parental sequence were introduced whenever deemed appropriate. The structural assessment was based on Fv region homology models of both the parental antibody and the humanization variants, created with an in-house antibody structure homology modeling protocol implemented using the BIO VIA Discovery Studio Environment, version 17R2. In some humanization variants, “forward mutations” were included, i.e., amino acid exchanges that change the original amino acid occurring at a given CDR position of the parental binder to the amino acid found at the equivalent position of the human acceptor germline. The aim was to increase the overall human character of the humanization variants (beyond the framework regions) to further reduce the immunogenicity risk.

[0413] All humanization variants include the C102S mutation (Kabat numbering) that eliminates a free cysteine in HCDR3 of the antibody as it was considered a developability risk.

[0414] An in silico tool developed in-house was used to predict the VH-VL domain orientation of the paired VH and VL humanization variants (WO 2016 / 062734, incorporated herein by reference in its entirety). The results were compared to the predicted VH-VL domain orientation of the parental binder to select for framework combinations which are close in geometry to the original antibodies. The rationale was to detect possible amino acid exchanges in the VH-VL interface region that might lead to disruptive changes in the pairing of the two domains that in turn might have detrimental effects on the binding properties.

[0415] Additionally, a crystal structure of the Fab fragment of GO2 bound to the peptide RPAPG- S(GalNAc)-T(GalNAc)-APPA (SEQ ID NO: 78) was solved and guided some refinements of the humanized variants (Figure 2).

[0416] The heavy and light chain vectors containing each of the humanized variants were cotransfected into Expi293™ suspension cells in a matrix manner to obtain cell cultures expressing full size humanized IgG of all possible light / heavy chain combinations. Cell culture supernatants were harvested and processed as described to obtain pure IgG preparations.

[0417] For a first series of humanized GO2 IgGs, four humanized VH variants and three humanized VL variants were designed (Table 1) and combined (Table 2). In addition, the free cysteine 102 in HCDR3 was mutated to serine in the otherwise unaltered GO2 VH. This mutated GO2 VH was combined with the unaltered GO2 VL to specifically investigate the impact of the C102S mutation on expression and binding.

[0418] The total IgG yields from 17 mL suspension culture were unexpectedly low, ranging from 26 to 225 pg for the humanized variants (a yield of 1-2 mg IgG typically being expected from this type of expression culture). Thus, all humanized GO2 variants were poorly expressed. The expression of the GO2 C102S mutant (P1AE6094) was only slightly higher.

[0419] Table 1. Overview of the humanized VH and VL domains of murine IgG GO2 that were part of the first series of humanized GO2 IgG variants. The respective germline sequences that were used for CDR grafting as well as introduced mutations are indicated. The original murine GO2 VH and VL domains are also listed together with a single amino acid mutant of VH.

[0420] *Back mutations are prefixed with "b", forward mutations with "f; residue indices given in

[0421] Kabat numbering

[0422] Table 2. Overview of the first series of humanized and chimeric GO2 variants. The MAb IDs are assigned to their respective VH and VL domains. The obtained small-scale production yields are also indicated.

[0423]

[0424] Next we tested the binding of the GO2 variants to T3M4 COSMC KO target cells using flow cytometry (Figure 3). In comparison to the parental GO2 TCB (P1AD8341 in Figure 3A) or GO2 IgG (Pl AA9754 in Figure 3B), binding was reduced, albeit to different extents, with all but one variant (P1AE6110).

[0425] In addition, we applied SPR to investigate in a qualitative manner whether the variants are still specific for the Tn-glycosylated epitope. For this purpose, we used the Tn-glycosylated target peptide Biotin-(O2Oc)2-RPAPG-S(GalNAc)-T(GalNAc)-APPAHGVT-amid (SEQ ID NO: 79) and its non-glycosylated analogue Biotin-(O2Oc)2-RPAPGSTAPPAHGVT-amid (SEQ ID NO: 80) as ligands.

[0426] All humanized variants (except P1AE6094) bound to the Tn-glycosylated peptide, but none bound to the unglycosylated peptide, indicating that the specificity for Tn-glycosylation was preserved after humanization. However variants comprising I-VHlb or I-VHld showed faster off-rates than variants comprising I-VH2a or I-VH2c (Figure 4).

[0427] In order to increase the expression yields, we created another series of humanized GO2 IgG variants. Since the X-ray-based structural analysis of the GO2 Fab suggested that the observed low productivity might be connected to the LCDR2, the germline sequence of the respective human VL gene was maintained in the newly designed humanized VL genes. As an additional control, the same procedure was applied to the murine VL domain of the chimeric GO2 IgG (Pl AA9754) (Table 3). Altogether, we designed five additional humanized VL domains. Three of them are based on human germline sequences that had been part of the first series already. In addition, one other human VL germline (IGKV1-39*O1) was evaluated. The five new VL variants were combined with four humanized VH domains. One of the four VH variants, I- VH2a, had been part of the first series already. Three additional ones are based on the same human germline sequence as I-VH2a (IGHV1-46*O1) but contain additional back and forward mutations (Table 3). In addition, each humanized VH and VL domain was combined with the murine GO2 VL or VH domain, respectively, for creating semi-humanized antibodies and further dissecting the impact of VH and VL on recombinant expression and target binding.

[0428] Table 3. Overview of the humanized and VH and VL domains of murine IgG GO2 that were part of the second series of humanized GO2 IgG variants. The respective germline sequences that were used for CDR grafting as well as introduced mutations are indicated.

[0429] *Back mutations are prefixed with "b", forward mutations with "f; residue indices given in Kabat numbering

[0430] +From the first series of humanized GO2 IgG variants

[0431] The resulting chimeric, semi-humanized and humanized IgGs were expressed in HEK293 cells as described and tested for binding to MCF7 COSMC KO cells using flow cytometry (Table 4).

[0432] Table 4. Overview of the second series of humanized GO2 variants. The semi-humanized and chimeric GO2 derivates as well as MAb P1AE6110 from the first series of humanized variants, which were used as controls, are also listed. The MAb IDs are assigned to their respective VH and VL domains. The obtained small-scale production yields and EC50 values for target cell binding are also indicated.

[0433]

[0434] The VH mutation C102S had no impact on either expression or target cell binding (compare MAb IDs P1AA9754 and P1AE6094). Replacing the LCDR2 of the chimeric GO2 IgG by the respective germline sequence increased the expression yield by more than a factor of three but also strongly reduced the binding to the target cells as indicated by the EC50 value of the flowcytometric binding assay (compare MAb IDs P1AA9754 and P1AF0261). In line with this, all semi-humanized variants comprising the original VL showed low to very low expression yields irrespective of which humanized VH was used (MAb IDs P1AF0255, P1AF0269, P1AF0270, P1AF0271). For those variants that were sufficiently expressed to enable the binding assay, EC50 values remained unchanged compared to the chimeric GO2 IgG (MAb IDs P1AF0269 and P1AF0271). Replacing the original VL by one of the humanized VLs led to a strong increase of expression but also to a decrease of target cell binding (Figure 5). This was true for combinations with the original VH and all humanized VH variants. In general, variants comprising VL2a_L2GL or VL3a_L2GL have the highest expression and the lowest EC50 values compared to other variants with the same VH. Among all fully humanized variants the combination of II-VH2d with either VL2a_L2GL or VL3a_L2GL (MAb IDs P1AF0291 and P1AF0294, bold in Table 4) yielded the highest expression (four to five-fold increase compared to the chimeric GO2 IgG) and the strongest target cell binding.

[0435] Next, we aimed at completely restoring target binding while maintaining high expression. For this purpose we designed another group of variants combining sequence elements of Pl AE6110 (humanized candidate showing strong target binding but poor expression) and P1AF0294 (humanized candidate showing the highest expression levels and only a moderate decrease in target binding). This approach was facilitated by the fact that P1AE6110 and P1AF0294 had been designed using the same human VH and VL germline sequences (IGHV1 -46*01 and IGKV2-28*01). Based on our previous analysis of the second group of humanized IgGs, as well as structural analysis of the GO2 Fab, we decided to keep the VH of P1AF0294 (II-VH2d) constant and focus on hybrids of the VL domains of P1AE6110 and P1AF0294 (Table 5).

[0436] Table 5. Overview of the humanized VL domains of murine IgG GO2 that were part of the third series of humanized GO2 IgG variants. The respective germline sequences that were used for CDR grafting as well as introduced mutations are indicated. *Back mutations are prefixed with "b", forward mutations with "f"; residue indices given in Kabat numbering

[0437] Additional VL variants were designed by backmutation to the murine germline sequence. The differences in the amino acid sequences of the new VL variants as well as the parental VLs of P1AE6110, P1AF0294 and GO2 (P1AA9754) together with the murine germline sequence IGKV8-30*01 are shown in Table 6.

[0438] The new LCs were combined with the HC of P1AF0294 (II-VH2d) and transiently expressed in 300 mL HEK suspension culture. Antibodies were purified and tested for binding to the Tn- glycosylated target peptide Biotin-(O2Oc)2-RPAPG-S(GalNAc)-T(GalNAc)-APPAHGVT- amid (SEQ ID NO: 79) as well as the non-glycosylated (SEQ ID NO: 80) or core- 1 -glycosylated (SEQ ID NO: 81) homologues. The chimeric GO2 IgG as well as P1AE6110 and P1AF0294 were tested for comparison. Table 6 summarizes the expression yields of the new variants as well as a qualitative assessment of their target binding. The corresponding sensorgrams are shown in Figure 6.

[0439] Among the new variants, only those carrying glycine instead of valine at position 51 (Kabat numbering) were expressed with acceptable yields ranging from 75 to 100 mg. Variants carrying either valine (P1AF2575), which represents the original amino acid of GO2, or alanine (P1AF2578), which is the amino acid in the corresponding murine VL germline, were poorly expressed. This suggests that G51 instead of V in LCDR2 is key to improving the expression of humanized GO2 variants. As for target binding, reintroduction of lysine at position 55 (Kabat numbering) of the VL of P1AF0294 was sufficient to restore the original target binding properties of GO2 (compare Pl AF0294 with P1AF2574 and Pl AA9754 in Table 6 and Figure 6).

[0440] Replacing the asparagine on position 53 (Kabat numbering) of Pl AF2574 by the corresponding threonine of the murine germline sequence, leading to P1AF2577, did neither improve the expression yield nor the target binding.

[0441] Neither GO2 nor any of the tested humanized variants bound to the non-glycosylated or core- 1 -glycosylated peptide (data not shown).

[0442] able 6. Production yields and qualitative target binding assessment of the third series of humanized GO2 antibodies contextualized with their CDRL2 equences. The GO2 chimeric antibody as well as MAbs P1AE6110 and P1AF0294 from the previous series of humanized antibodies, which were sed as controls in the SPR experiment, are also listed. The corresponding sequence stretch of the murine germline homologue of GO2 is indicated or comparison.

[0443] Example 3 - Generation of affinity matured humanized GO2 Fab variants

[0444] Library design

[0445] The humanized GO2 IgG P1AF0294 was subjected to affinity maturation by phage display of a synthetic Fab library. In order to guide this process we analyzed a crystal structure of antibody P1AF2574 in complex with a MUC-1 derived peptide RPAPG-S(GalNAc)-T(GalNAc)-APPA (SEQ ID NO: 78) carrying two O-linked glycosylation sites of the aberrant Tn-type. An interaction map was generated between the antibody and the Tn-type carbohydrate structure of the antigen, as well as between the antibody and the peptide backbone of the antigen. We aimed to increase the affinity preferentially to the carbohydrate antigen while keeping the affinity to the peptide unchanged. This was considered useful since tumor specificity should be achieved via targeting the Tn-type carbohydrate antigen.

[0446] A phage displayed library was generated using the template antibody sequence. Randomization of the library was such that residues within the appropriate distance to the Tn-moiety of the antigen were replaced by amino acids with hydrogen bond donor or acceptor properties, thus facilitating an optimized hydrogen bonding network around the carbohydrate. By this, we aimed to increase the specificity for this particular carbohydrate moiety. In total 3 positions in VL and 14 positions in VH were randomized (Table 7). Phage particles displaying the PlAF0294-derived affinity maturation library were produced using standard procedures and used for selection.

[0447] Table 7. Randomization of the amino acid sequences of Mab P1AF0294 VH and VL domains for affinity maturation. Amino acids used for randomization are assigned to the respective amino acid positions of the template. The amino acids that are present in the template sequence are also indicated.

[0448] Selection of affinity-matured P lAF0294-derived phage clones

[0449] For the identification of affinity-matured clones, phage display selection was performed using a biotinylated 40-mer peptide harboring two copies of the MUC-1 repeat sequence with Tn glycosylation (Tn-40mer, SEQ ID NO: 82; see also Table 8). Panning rounds were performed in solution according to the following pattern:

[0450] 1. Pre-clearing of non-specific phagemid particles by incubation with 250 nM biotinylated MUC-1 40-mer harboring two copies of core- 1 -glycosylated MUC-1 repeats (core-l-40mer, SEQ ID NO: 84) and the corresponding biotinylated, non-glycosylated MUC-1 40-mer (non- glyco-40mer, SEQ ID NO: 83) for Ih.

[0451] 2. Capturing of biotinylated peptides and bound phagemid particles by addition of 5.4 x 107streptavidin-coated magnetic beads for 10 min.

[0452] 3. Isolation of non-bound phagemid particles from supernatant for further selection.

[0453] 4. Binding of phagemid particles to biotinylated Tn-40mer for 0.5 h in a total volume of 1 ml at concentrations according to the panning strategy (Table 9).

[0454] 5. Capturing of biotinylated Tn-40mer and specifically bound phage particles by addition of 5.4 x 107streptavidin-coated magnetic beads for 10 min.

[0455] 6. Washing of beads using 5 x 1 ml PBS / Tween20 and 5 x 1 ml PBS.

[0456] 7. Elution of phage particles by addition of 1ml 100 mM TEA for 10 min and subsequent neutralization by adding 500 pl I M Tris / HCl pH 7.4.

[0457] 8. Infection of exponentially growing E. coli TGI bacteria with eluted phage and incubation for 30 min at 37°C.

[0458] 9. Infection with helper phage VCSM13 and incubation of the culture at 30°C overnight.

[0459] 10. Subsequent PEG / NaCl precipitation of phagemid particles to be used in subsequent selection rounds. Selections were carried out over 4 rounds using two different stringencies, but both with decreasing antigen concentrations (Table 9).

[0460] For the screening of specific and affinity -matured binders, single colonies of phage- transfected bacteria were inoculated in 1 ml bacterial media. At an OD600 of about 0.5-0.6, phagemid- derived Fab expression was induced by adding 1 mM isopropyl- P-D-l -thiogalactopyranoside (IPTG) to the culture. After incubation at 30°C overnight, bacteria were spun down and the bacterial supernatant, which contained the soluble Fab fragments, was harvested. Specific binders were identified using the plate-based fluorescence cytometer Mirrorball. Table 8. List of MUC-1 peptides used for crystallization, assessment of target binding, phage pre-clearing or panning.

[0461] Table 9. Overview of conditions for phage pre-clearing and panning.

[0462] Identification of affinity-matured P lAF0294-derived variants by Mirrorball

[0463] For the identification of affinity-matured binder variants of P1AF0294 that are specific for Tn- MUC-1 but do not bind to core-1 nor non-glycosylated MUC-1, the plate-based fluorescence cytometer Mirrorball (SPT Labtech) was used.

[0464] Each of the three biotinylated MUC-1 40-mer antigens and a biotinylated antibody Fc fragment were coated to specific streptavidin-beads with different fluorescence emission spectra. This approach allows the simultaneous binding analysis to all antigens and the control Fc in the same well.

[0465] In a first step, 10 pl of each type of beads (of sol-R2, sol-R3, sol-R4 and sol-R5 beads) were washed separately with 1 ml PBS and centrifuged for 5 min at 14000 x g. After removal of the washing solution, the beads were resuspended in 1 ml PBS containing 80 nM of the respective antigen. After incubation for 1 h at RT with gentle rotation, the beads were again centrifuged for 5 min at 14000 x g, once washed with 1 ml PBS, and finally resuspended in 14 ml PBS.

[0466] A secondary FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, # 109-096-097) was added to the beads at a concentration of 800 ng / ml. Using a 384 microtiter plate, 35 pl of the resulting solution were then pipetted to each well and combined with 5 pl of bacterial supernatant containing potentially affinity-matured Fab clones of P1AF0294. After an incubation time of 1 h at RT, the plate was analyzed by the Mirrorball device. Clones that were only fluorescence-positive for the Tn-40mer (SEQ ID NO: 82) but negative for either of the control antigens were selected and sequenced.

[0467] DNA Sequence analysis of 320 clones revealed 47 non-redundant pairs of VL and VH. However, it became obvious that the sequence of the library template P1AF0294 had been conserved in almost all positions. Slight preferences for the amino acid replacements Y96W and D99K or R in VH as well as L50W in VL (Kabat numbering) were the only exceptions from this observation. F100Y in VH and G51V in VL (Kabat numbering) were tolerated and observed at about the same rate as they were represented in the library. Example 4 - Preparation of GO2-derived humanized Tn-MUC-1 CD3 T-cell bispecific (TCB) antibodies

[0468] A further optimization of humanized GO2-derived Tn-MUC-1 binders was performed in the TCB antibody format by systematically combining the humanized VH and VL sequences of antibody P1AF2574 with amino acid substitutions that had been identified as preferred or at least tolerated by phage display.

[0469] “CD30rig” was used as the CD3 binder in this example. The VH and VL sequences of this binder are provided in the sequence listing (SEQ ID NOs 67 and 68, respectively).

[0470] Exemplary sequences of such a TCB are given in SEQ ID NOs 69-72 (Molecule P1AG1650). Other TCB antibodies were constructed in an analogous manner, using the VH and VL sequences of the corresponding humanized GO2-derived Tn-MUC-1 binder. Table 10 shows an overview of the sequence properties of the humanized GO2-derived TCBs together with the production yields from a IL HEK suspension culture. In line with what was observed with the corresponding antibodies, variants containing VL V51 were poorly expressed (P1AG1651, P1AG1652, P1AG1654 and P1AG1659). Replacing V51 with G greatly improved expression yields (compare P1AG1651 with P1AG1650, P1AG1652 with P1AG5620, P1AG5654 with P1AG5621, and P1AG1659 with P1AG1655). This was observed after the initial affinity chromatography as well as with the fully purified samples.

[0471] able 10. Overview of TCBs that where designed by combining the humanized VH and VL sequences of antibody P1AF2574 with amino acid ubstitutions that had been identified by phage display-based affinity maturation. Amino acids at relevant positions are indicated, with amino acids eviating from the P1AF0294 sequence highlighted with bold letters. Purification yields after the initial protein A-affinity chromatography (AFC) as ell as the final yields are indicated. Samples were purified by either a three-column process (AFC, SEC and cIEX) or a two-column process (AFC nd SEC; see asterisks).

[0472] two step purification with AFC and SEC

[0473] Example 5 - Testing of GO2-derived humanized Tn-MUC-1 CD3 T-cell bispecific (TCB) antibodies

[0474] The bispecific constructs were tested to confirm that target cell binding was preserved. Indeed, as shown exemplary for two TCBs in Figure 7, target binding properties of the TCBs were comparable to the control. Next, T cell mediated tumor cell killing was assessed. Figure 8 depicts results from a T cell mediated tumor cell killing assay and T cell activation measurement performed using MCF7 COSMC KO. PBMCs were used as effector cells to assess target cell killing capacity upon T cell activation. Selected variants showed lower EC50 values as compared to the GO2 TCB (P1AD8341). Corresponding EC50 values are shown in Table 11. Table 11. Summary of EC50 values from the T cell mediated tumor cell killing and T cell activation.

[0475] Table 12. Summary of EC50 values from the T cell mediated tumor cell killing and T cell activation for two additional TCBs in comparison with GO2 TCB (P1AD8341).

[0476] Lack of off-target activity was tested with HBEpiC cells isolated from human bronchi (ScienCell Research Laboratories, #3210). Except TCB P1AG1652 and P1AG1654, all TCBs showed an equal or improved profile (equal or reduced activity towards HBEpiC cells) compared to GO2 TCB P1AD8341 as illustrated in Figure 9A showing T-cell mediated cell killing and Figure 9B showing the percentage of CD25+ cells out of CD4+ T cells.

[0477] In a separate experiment, we assessed target cell killing and off-target activity on MCF7 CO SMC KO cells and on HBEpiC with Pl AG5620 and Pl AG5621 compared to the GO2 TCB P1AD8341 (Figure 10). The TCBs showed comparable potency on target cells (Table 12). However, off- target activity was observed with P1AG5621.

[0478] In addition, we assessed TCB potency of P1AG1650 and P1AG1655 in a target cell killing assay by looking at cumulative cell death over the course of 4 days (Figure 11). The GO2 (P1AD8341, VH SEQ ID NO: 1 / VL SEQ ID NO: 7; see also WO 2019 / 083506) and 4AG (P1AE4887, VH SEQ ID NO: 34 / VL SEQ ID NO: 35; see also WO 2020 / 006449) TCBs were included as a reference. Superior potency of the GO2-derived molecules was observed compared to the parental molecule P1AD8341 as well as the 4AG TCB.

[0479] The anti-Tn-MUC-1 variable domains of TCB P1AG1650 (SEQ ID NOs 27 and 31) were crafted to the human gamma 1 constant region containing the PGLALA mutation or the wild-typ kappa constant region, respectively, yielding IgG P1AH6390. In the same way the variable domains of TCB P1AG1655 (SEQ ID NOs 13 and 20) were used to create IgG P1AF2574. The IgGs P1AH6390 and P1AF2574 were tested for their specificity towards Tn-glycosylation in comparison to the parental GO2 IgGPlAA9754. To this end, flow cytometry was performed with HEK 293A COSMC KO + MUC1 cells, which overexpress MUC-1 and are positive for Tn glycosylation, and HEK 293 A + MUC1 cells, which overexpress MUC-1 but are negative for Tn glycosylation.

[0480] P1AH6390 (Figure 12A), P1AF2574 (Figure 12B), and P1AA9754 (Figure 12C) all bind to the Tn-positive cell line but not to the Tn-negative cell line. These results confirm the strong Tn-glycosylation specificity of the parental GO2 IgG as well as of the humanized variants P1AH6390 and P1AF2574.

[0481] The monovalent affinities of IgGs P1AH6390 and P1AF2574 were determined by SPR using the corresponding Fab fragments as analyte and biotinylated Tn-MUC-1 peptide (SEQ ID NO: 79) as ligand. The Fab of the parental GO2 IgG P1AA9754 was analyzed for comparison. The corresponding KD values are listed in Table 13.

[0482] Table 13. Monovalent affinities of anti-Tn-MUC-1 antibodies as compared to parental GO2.

[0483] * * *

[0484] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

-98-CLAIMS1. An antibody that binds to Tn-MUC-1, wherein the antibody is a humanized antibody comprising a heavy chain variable region (VHMUC-I) and a light chain variable region (VLMUC-I) derived from a parental antibody, the parental antibody comprising the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 7, wherein the VLMUC- i comprises glycine at position 51 (Kabat numbering).

2. The antibody of claim 1, wherein the VLMUC-I comprises lysine at position 55 (Kabat numbering).

3. The antibody of claim 1 or 2, wherein the VHMUC-I comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 45, and the VLMUC-I comprises the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 53 and the LCDR 3 of SEQ ID NO: 56.

4. An antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable region (VHMUC-I) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 36, the HCDR 2 of SEQ ID NO: 38, and the HCDR 3 of SEQ ID NO: 45, and a light chain variable region (VLMUC-I) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 48, the LCDR 2 of SEQ ID NO: 53 and the LCDR 3 of SEQ ID NO: 56.

5. The antibody of any one of the preceding claims, wherein the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and a HCDR3 selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, a LCDR2 selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56.

6. The antibody of any one of the preceding claims, wherein(i) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56;(ii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;-99-(iii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 41; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;(iv) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 42; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;(v) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 43; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 51, and the LCDR3 of SEQ ID NO: 56;(vi) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56;(vii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 44; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 52, and the LCDR3 of SEQ ID NO: 56; or(viii) the VHMUC-I comprises the HCDR1 of SEQ ID NO: 36, the HCDR2 of SEQ ID NO: 38, and the HCDR3 of SEQ ID NO: 40; and the VLMUC-I comprises the LCDR1 of SEQ ID NO: 48, the LCDR2 of SEQ ID NO: 50, and the LCDR3 of SEQ ID NO: 56.

7. The antibody of any one of the preceding claims, wherein the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 65; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66.

8. The antibody of any one of the preceding claims, wherein the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 31.

9. The antibody of any one of the preceding claims, wherein(i) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I-100- comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31;(ii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;(iii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;(iv) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;(v) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20;(vi) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31;(vii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31; or(viii) the VHMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13 and / or the VLMUC-I comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

10. The antibody of any one of claims 1 to 9, wherein the antibody is an antibody fragment, optionally selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab’)2 molecule.-101-11. The antibody of any one of claims 1 to 9, wherein the antibody comprises an Fc region, particularly an IgGFc region, more particularly an IgGi Fc region, most particularly a human IgGi Fc region.

12. The antibody of any one of claims 1 to 9, wherein the antibody is a full-length antibody particularly a full-length IgG antibody, more particularly a full-length IgGi antibody, most particularly a full-length human IgGi antibody.

13. The antibody of any one of claims 1 to 12, wherein the antibody is a multispecific antibody, particularly a bispecific antibody, optionally wherein the multispecific antibody binds to Tn-MUC- 1 and to CD3.

14. Isolated polynucleotide encoding the antibody of any one of claims 1 to 13.

15. A host cell comprising the polynucleotide of claim 14.

16. A method of producing an antibody that binds to Tn-MUC-1, comprising culturing the host cell of claim 15 under conditions suitable for the expression of the antibody, and optionally further comprising recovering the antibody from the host cell.

17. An antibody that binds to Tn-MUC-1 produced by the method of claim 16.

18. A pharmaceutical composition comprising the antibody of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

19. The antibody of any one of claims 1 to 13 or the pharmaceutical composition of claim 18 for use as a medicament.

20. The antibody of any one of claims 1 to 13 or the pharmaceutical composition of claim 18 for use in the treatment of cancer.

21. Use of the antibody of any one of claims 1 to 13 or the pharmaceutical composition of claim 18 in the manufacture of a medicament.

22. Use of the antibody of any one of claims 1 to 13 or the pharmaceutical composition of claim 18 in the manufacture of a medicament for the treatment of cancer.

23. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the antibody of any one of claims 1 to 13 or the pharmaceutical composition of claim 18.The invention as described hereinbefore.

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