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 P39420

[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 / 19.11.2025 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 pharmaceutical development and therapeutic purposes. In particular, the antibodies provide specific binding to Tn-MUC-1, as opposed to normally glycosylated MUC-1 or Tn-antigens other than Tn-MUC-1. The antibodies also provide species crossreactivity for human and cynomolgus Tn-MUC-1, as well as good functional activity.

[0013] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises (i) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; or (ii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0014] In one aspect, (i) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VH sequence of SEQ ID NO: 4; and / or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VL sequence of SEQ ID NO: 8; or (ii) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VH sequence of SEQ ID NO: 12; and / or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VL sequence of SEQ ID NO: 16. In one aspect, (i) the VH comprises the amino acid sequence of SEQ ID NO: 4 and the VL comprises the amino acid sequence of SEQ ID NO: 8; or (ii) the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO: 16.

[0015] In a further aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO:8; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0016] In one aspect, the antibody of the invention 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. In another aspect, the antibody comprises an Fc region, particularly an IgG Fc region, more particularly an IgGi Fc region, most particularly a human IgGi Fc region. In still another aspect, 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. In one aspect, the antibody is a multispecific antibody, particularly a bispecific antibody, optionally wherein the multispecific antibody binds to Tn-MUC-1 and to CD3.

[0017] 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.

[0018] 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.

[0019] The invention further provides a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically acceptable carrier.

[0020] 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. 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.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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” with charge modifications (VH / VL exchange in T-cell receptor binder; charge modifications in Tn- MUC-1 binders, EE = 147E, 213E; RK = 123R, 124K).

[0023] Figure 2. Activation of anti-P329G chimeric antigen receptor (CAR) transduced, COSMC overexpressing Jurkat-NFAT-luciferase reporter cells (P329G-CAR-J COSMC). Target specific activation was determined with MCF7 COSMC KO or MCF7 COSMC K0-MUC1 KO cells. Off-target activity was assessed on HBEpiC cells. Cells were exposed to anti-Tn-MUC- 1 IgGs containing the P239G mutation at various concentrations for 24 hours and reporter gene activation was measured by luminescence using OneGlo reagent (Promega).

[0024] Figure 3. Target cell killing of MCF7 COSMC KO cells and off-target activity on HBEpiC cells. PBMCs from a healthy donor were exposed to Tn-MUC-1 TCBs at various concentrations and co-incubated with target or off-target cells for 72 hours. Cell killing was measured by quantification of cell death using CytotoxGlo reagent (Promega).

[0025] Figure 4. Target cell killing of MCF7 COSMC KO or COSMC K0-MUC1 KO cells and off- target activity on HBEpiC. PBMCs from a healthy donor were exposed to Tn-MUC-1 TCBs at various concentrations and co-incubated with target or off-target cells for 72 hours. Cell killing was measured by quantification of cell death using CytotoxGlo reagent (Promega).

[0026] Figure 5. Dose-dependent binding of IgGs P1AG0639 (B) and P1AG0648 (C) to a panel of HEK 293A-derived engineered cell lines in comparison to GO2 IgG (P1AA9754) (A) as measured by flow cytometry. Antibodies bound to target cells were detected with a fluorescently labeled anti-human Fc specific secondary antibody. Figure 6. Dose-dependent binding of TCBs Pl AG5361 and Pl AG5364 to HEK 293 A COSMC KO cells either overexpressing human (A) or cynomolgus (B) MUC-1 as measured by flow cytometry. Antibodies bound to target cells were detected with a fluorescently labeled antihuman Fc specific secondary antibody.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] I. DEFINITIONS

[0029] 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 one aspect, 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: 25):

[0030] 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.

[0031] 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 one aspect, 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 one aspect, an antibody that binds to Tn-MUC-1 has a dissociation constant (KD) of < 1 pM, < 500 nM, < 250 nM, < 200 nM, < 100 nM, < 50 nM, < 25 nM, < 20 nM, < 10 nM, < 1 nM, or < 0.1 nM, particularly < 500 nM, more particularly < 250 nM, most particularly

[0032] < 200 nM.

[0033] 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 one aspect, 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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 one aspect, a human antibody is derived from a non-human transgenic mammal, for example a mouse, a rat, or a rabbit. In one aspect, 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.

[0038] 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, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0039] “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).

[0040] 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).

[0041] 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 one aspect, 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. 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.

[0042] An ’’isolated” antibody is one which has been separated from a component of its natural environment. In one aspect, 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 one aspect, the antibody provided by the present invention is an isolated antibody.

[0043] 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).

[0044] 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.

[0045] 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, 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”).

[0046] 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.

[0047] 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): (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”);

[0048] (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 1991 (“Kabat definition”);

[0049] (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

[0050] (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”).

[0051] Unless otherwise indicated, the CDRs are determined herein according to 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 definition / system.

[0052] ’’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-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.

[0053] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat 1991.

[0054] 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?), y? (IgG?), 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.

[0055] 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.

[0056] 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 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.

[0057] “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.

[0058] “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).

[0059] 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.

[0060] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to one or more cytotoxic agent(s).

[0061] 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, non-conservative 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.

[0062] “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 pursposes 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] “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.

[0067] 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”.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 one aspect, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease. 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.

[0072] 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.

[0073] 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.

[0074] II. COMPOSITIONS AND METHODS

[0075] The invention provides antibodies that bind Tn-MUC-1. The antibodies show favorable properties for pharmaceutical development and therapeutic application, including specific binding to Tn-MUC-1 (as opposed to normally glycosylated MUC-1 or Tn-antigens other than Tn-MUC-1), species cross-reactivity between human and cynomolgous Tn-MUC-1, as well as good functional activity. Antibodies of the invention as useful, e.g., for the treatment of diseases such as cancer.

[0076] A. Anti-Tn-MUC-1 antibodies

[0077] 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.

[0078] P1AG0639

[0079] In one aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2.

[0080] In one aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0081] In another aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 7.

[0082] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7.

[0083] In one aspect, the antibody is an isolated antibody and / or a purified antibody. In one aspect, the antibody is a monoclonal antibody. In one aspect, the antibody is a humanized or human antibody.

[0084] In one aspect, the antibody has any one more more of the following properties or functions: selectively binds to Tn-MUC-1 (in particular binds to Tn-MUC-1, but not other glycoforms of MUC-1 or Tn-antigens other than Tn-MUC-1); binds to the APPAHGV-T(GalNAc)-S(GalNAc)-APD (SEQ ID NO: 26) epitope of Tn- MUC-1; binds to both human and cynomolgus Tn-MUC-1; and / or binds to (human) Tn-MUC-1 with an affinity of < 500 nM, particularly < 250 nM, more particularly < 200 nM as measured by SPR at 25°C.

[0085] In one aspect, the antibody further comprises a human framework, e.g. a human immunoglobulin framework or a human consensus framework.

[0086] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0087] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, the anti-Tn- MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0088] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence.

[0089] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising the amino acid sequence of SEQ ID NON comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising the amino acid sequence of SEQ ID NON comprising one amino acid substitution within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising two amino acid substitutions within (the framework region of) said amino acid sequence.

[0090] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0091] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, the anti-Tn- MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0092] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence.

[0093] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising one amino acid substitution within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising two amino acid substitutions within (the framework region of) said amino acid sequence.

[0094] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4; and (ii) at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0095] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VH having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4; and (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and a VL having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, the VH has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, the VL has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, the VH has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, the VL has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0096] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence; and (ii) at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence.

[0097] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence; and (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence. In one aspect, the VH comprises one amino acid substitution within the amino acid sequence of SEQ ID NO: 4. In one aspect, the VH comprises two amino acid substitutions within the amino acid sequence of SEQ ID NO: 4. In one aspect, the VL comprises one amino acid substitution within the amino acid sequence of SEQ ID NO: 8. In one aspect, the VL comprises two amino acid substitutions within the amino acid sequence of SEQ ID NO: 8.

[0098] In another aspect, the anti-Tn-MUC-1 antibody comprises one or more of the CDR amino acid sequences of the VH of SEQ ID NO: 4, and one of more of the CDR amino acid sequences of the VL of SEQ ID NO: 8

[0099] In a further aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH of SEQ ID NO: 4, and the CDR-L1, CDR-L2 and CDR- L3 amino acid sequences of the VL of SEQ ID NO: 8.

[0100] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 4 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 4.

[0101] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 4. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and a heavy chain FR amino acid sequence of at least 95% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 4. In another aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and a heavy chain FR amino acid sequence of at least 98% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 4.

[0102] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 4 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence.

[0103] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR- Hl, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and the heavy chain FR amino acid sequence of the VH of SEQ ID NON comprising one amino acid substitution within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and the heavy chain FR amino acid sequence of the VH domain of SEQ ID NO: 4 comprising two amino acid substitutions within said FR amino acid sequence.

[0104] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 8 and a light chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 8.

[0105] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and a light chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and a light chain FR amino acid sequence of at least 95% sequence identity to the FR amino acid sequence of the VL domain of SEQ ID NO: 8. In another aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and a light chain FR amino acid sequence of at least 98% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 8 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence.

[0106] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR- Ll, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 8 comprising one amino acid substitution within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 8 comprising two amino acid substitutions within said FR amino acid sequence.

[0107] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 4 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 4, and (ii) one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 8 and a light chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises (i) the CDR- Hl, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 4, and (ii) the CDR-L1, CDR- L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and a light chain FR amino acid sequence of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 8. In one aspect, the heavy chain FR amino acid sequence has at least 95% sequence identity to the FR amino acid sequence of SEQ ID NO: 4. In one aspect, the light chain FR amino acid sequence has at least 95% sequence identity to the FR amino acid sequence of SEQ ID NO: 8. In one aspect, the heavy chain FR amino acid sequence has at least 98% sequence identity to the FR amino acid sequence of SEQ ID NO: 4. In one aspect, the light chain FR amino acid sequence has at least 98% sequence identity to the FR amino acid sequence of SEQ ID NO: 8.

[0108] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 4 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence, and (ii) one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 8 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence.

[0109] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 4 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence, and (ii) the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 8 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence. In one aspect, the heavy chain FR sequence comprises one amino acid substitution within the FR amino acid sequence of SEQ ID NO: 4. In one aspect, the heavy chain FR sequence comprises two amino acid substitutions within the FR amino acid sequence of SEQ ID NO: 4. In one aspect, the light chain FR sequence comprises one amino acid substitution within the FR amino acid sequence of SEQ ID NO: 8. In one aspect, the light chain FR sequence comprises two amino acid substitutions within the FR amino acid sequence of SEQ ID NO: 8.

[0110] Preferably, the CDRs of the VH and / or VL according to the above aspects are according to the Kabat definition. Alternatively, the CDRs of the VH and / or VL according to the above aspects are according to the Chothia definition. Further alternatively, the CDRs of the VH and / or VL according to the above aspects are according to the Contact definition. Still further alternatively, the CDRs of the VH and / or VL according to the above aspects are according to the IMGT definition.

[0111] Preferably, the antibody according to the above aspects binds to Tn-MUC-1 with a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0112] In a further aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, the anti-Tn-MUC-1 antibody comprises VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence SEQ ID NO: 4 contains substitutions (e.g., conservative substitutions), insertions, or deletions within SEQ ID NO: 4, wherein the anti-Tn-MUC-1 antibody retains the ability to bind to Tn-MUC-1. In one aspect, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 4. In one aspect, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0113] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising one amino acid substitution within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising two amino acid substitutions within said amino acid sequence. In one aspect, the substitutions are in the FR of the VH.

[0114] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4. Optionally, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0115] In a further aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising an amino acid sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8. In one aspect, an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence SEQ ID NO: 8 contains substitutions (e.g., conservative substitutions), insertions, or deletions within SEQ ID NO: 8, wherein the anti-Tn-MUC-1 antibody retains the ability to bind to Tn-MUC-1. In one aspect, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 8. In one aspect, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0116] In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising one amino acid substitution within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising two amino acid substitutions within said amino acid sequence. In one aspect, the substitutions are in the FR of the VL.

[0117] In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8. Optionally, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) CDR- L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7.

[0118] In another aspect, an anti-Tn-MUC-1 antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.

[0119] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the VH sequence of SEQ ID NO: 4, and a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the VL sequence of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the VH sequence of SEQ ID NO: 4, and a VL comprising an amino acid sequence having at least 95% sequence identity to the VL sequence of SEQ ID NO: 8. In one aspect, the anti-Tn- MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the VH sequence of SEQ ID NO: 4, and a VL comprising an amino acid sequence having at least 98% sequence identity to the VL sequence of SEQ ID NO: 8.

[0120] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence, and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising one amino acid substitution within said amino acid sequence, and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising one amino acid substitution within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 comprising two amino acid substitutions within said amino acid sequence, and a VL comprising the amino acid sequence of SEQ ID NO: 8 comprising two amino acid substitutions within said amino acid sequence. In one aspect, the substitutions are in the FR of the VH and VL.

[0121] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 8. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH and a VL comprising the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 8, respectively, including post-translational modifications of those sequences.

[0122] In one aspect, the anti-Tn-MUC-1 antibody comprises (a) a VH comprising an amino acid sequence having a single amino acid substitution in the VH sequence of SEQ ID NO: 4; and / or (b) a VL comprising an amino acid sequence having a single amino acid substitution in the VL sequence of SEQ ID NO: 8. In one aspect, the substitution is in the FR of the VH and / or VL. In one aspect, the anti-Tn-MUC-1 antibody comprises (a) a VH comprising an amino acid sequence having two amino acid substitutions in the VH sequence of SEQ ID NO: 4; and / or (b) a VL comprising an amino acid sequence having two amino acid substitutions in the VL sequence of SEQ ID NO: 8. In one aspect, the substitutions are in the FR of the VH and / or VL.

[0123] P1AG0648 In one aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In one aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In another aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In a further aspect, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10.

[0124] In one aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In a further aspect, the antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11.

[0125] In another aspect, the invention provides an antibody that binds to Tn-MUC-1 comprising at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In one aspect, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0126] In one aspect, the invention provides an antibody that binds to Tn-MUC-1, wherein the antibody comprises a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0127] In one aspect, the antibody is an isolated antibody and / or a purified antibody. In one aspect, the antibody is a monoclonal antibody. In one aspect, the antibody is a humanized or human antibody.

[0128] In one aspect, the antibody has any one more more of the following properties or functions: selectively binds to Tn-MUC-1 (in particular binds to Tn-MUC-1, but not other glycoforms of MUC-1 or Tn-antigens other than Tn-MUC-1); binds to the APPAHGV-T(GalNAc)-S(GalNAc)-APD (SEQ ID NO: 26) epitope of Tn- MUC-1; binds to both human and cynomolgus Tn-MUC-1; and / or binds to (human) Tn-MUC-1 with an affinity of < 500 nM, particularly < 200 nM, , more particularly < 200 nM as measured by SPR at 25°C.

[0129] In one aspect, the antibody further comprises a human framework, e.g. a human immunoglobulin framework or a human consensus framework.

[0130] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0131] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the anti- Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0132] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence.

[0133] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:

[0134] 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising one amino acid substitution within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising two amino acid substitutions within (the framework region of) said amino acid sequence.

[0135] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0136] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the anti- Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:

[0137] 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0138] In one aspect, the anti-Tn-MUC-1 antibody comprises at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence.

[0139] In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:

[0140] 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising one amino acid substitution within (the framework region of) said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising two amino acid substitutions within (the framework region of) said amino acid sequence.

[0141] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12; and (ii) at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0142] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12; and (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the VH has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the VL has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the VH has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the VL has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0143] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) at least one, at least two, or all three VH CDR sequences selected from (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence; and (ii) at least one, at least two, or all three VL CDR sequences selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence.

[0144] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence; and (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within (the framework region of) said amino acid sequence. In one aspect, the VH comprises one amino acid substitution within the amino acid sequence of SEQ ID NO: 12. In one aspect, the VH comprises two amino acid substitutions within the amino acid sequence of SEQ ID NO: 12. In one aspect, the VL comprises one amino acid substitution within the amino acid sequence of SEQ ID NO: 16. In one aspect, the VL comprises two amino acid substitutions within the amino acid sequence of SEQ ID NO: 16.

[0145] In another aspect, the anti-Tn-MUC-1 antibody comprises one or more of the CDR amino acid sequences of the VH of SEQ ID NO: 12, and one of more of the CDR amino acid sequences of the VL of SEQ ID NO: 16.

[0146] In a further aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH of SEQ ID NO: 12, and the CDR-L1, CDR-L2 and CDR- L3 amino acid sequences of the VL of SEQ ID NO: 16.

[0147] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 12 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 12.

[0148] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 12. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and a heavy chain FR amino acid sequence of at least 95% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 12. In another aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and a heavy chain FR amino acid sequence of at least 98% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 12.

[0149] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 12 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence.

[0150] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 12 comprising one amino acid substitution within said FR amino acid sequence. In one aspect, the anti-Tn- MUC-1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and the heavy chain FR amino acid sequence of the VH domain of SEQ ID NO: 12 comprising two amino acid substitutions within said FR amino acid sequence.

[0151] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 16 and a light chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 16.

[0152] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and a light chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and a light chain FR amino acid sequence of at least 95% sequence identity to the FR amino acid sequence of the VL domain of SEQ ID NO: 16. In another aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and a light chain FR amino acid sequence of at least 98% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 16.

[0153] In one aspect, the anti-Tn-MUC-1 antibody comprises one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 16 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence.

[0154] In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 16 comprising one amino acid substitution within said FR amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 16 comprising two amino acid substitutions within said FR amino acid sequence.

[0155] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 12 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 12, and (ii) one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 16 and a light chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises (i) the CDR- Hl, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and a heavy chain FR amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the FR amino acid sequence of the VH of SEQ ID NO: 12, and (ii) the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and a light chain FR amino acid sequence of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the FR amino acid sequence of the VL of SEQ ID NO: 16. In one aspect, the heavy chain FR amino acid sequence has at least 95% sequence identity to the FR amino acid sequence of SEQ ID NO: 12. In one aspect, the light chain FR amino acid sequence has at least 95% sequence identity to the FR amino acid sequence of SEQ ID NO: 16. In one aspect, the heavy chain FR amino acid sequence has at least 98% sequence identity to the FR amino acid sequence of SEQ ID NO: 12. In one aspect, the light chain FR amino acid sequence has at least 98% sequence identity to the FR amino acid sequence of SEQ ID NO: 16.

[0156] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) one or more of the heavy chain CDR amino acid sequences of the VH of SEQ ID NO: 12 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence, and (ii) one or more of the light chain CDR amino acid sequences of the VL of SEQ ID NO: 16 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence.

[0157] In one aspect, the anti-Tn-MUC-1 antibody comprises (i) the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH of SEQ ID NO: 12 and the heavy chain FR amino acid sequence of the VH of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence, and (ii) the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL of SEQ ID NO: 16 and the light chain FR amino acid sequence of the VL of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said FR amino acid sequence. In one aspect, the heavy chain FR sequence comprises one amino acid substitution within the FR amino acid sequence of SEQ ID NO: 12. In one aspect, the heavy chain FR sequence comprises two amino acid substitutions within the FR amino acid sequence of SEQ ID NO: 12. In one aspect, the light chain FR sequence comprises one amino acid substitution within the FR amino acid sequence of SEQ ID NO: 16. In one aspect, the light chain FR sequence comprises two amino acid substitutions within the FR amino acid sequence of SEQ ID NO: 16.

[0158] Preferably, the CDRs of the VH and / or VL according to the above aspects are according to the Kabat definition. Alternatively, the CDRs of the VH and / or VL according to the above aspects are according to the Chothia definition. Further alternatively, the CDRs of the VH and / or VL according to the above aspects are according to the Contact definition. Still further alternatively, the CDRs of the VH and / or VL according to the above aspects are according to the IMGT definition.

[0159] Preferably, the antibody according to the above aspects binds to Tn-MUC-1 with a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0160] In a further aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 12. In one aspect, the anti-Tn-MUC-1 antibody comprises VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one aspect, an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence SEQ ID NO: 12 contains substitutions (e.g., conservative substitutions), insertions, or deletions within SEQ ID NO: 12, wherein the anti-Tn-MUC-1 antibody retains the ability to bind to Tn-MUC-1. In one aspect, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 12. In one aspect, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0161] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising one amino acid substitution within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising two amino acid substitutions within said amino acid sequence. In one aspect, the substitutions are in the FR of the VH.

[0162] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12. Optionally, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11.

[0163] In a further aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising an amino acid sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence SEQ ID NO: 16 contains substitutions (e.g., conservative substitutions), insertions, or deletions within SEQ ID NO: 16, wherein the anti-Tn-MUC-1 antibody retains the ability to bind to Tn-MUC-1. In one aspect, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 16. In one aspect, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0164] In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising one amino acid substitution within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising two amino acid substitutions within said amino acid sequence. In one aspect, the substitutions are in the FR of the VL.

[0165] In one aspect, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 16. Optionally, the anti-Tn-MUC-1 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (b) CDR- L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In another aspect, an anti-Tn-MUC-1 antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.

[0166] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the VH sequence of SEQ ID NO: 12, and a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the VL sequence of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the VH sequence of SEQ ID NO: 12, and a VL comprising an amino acid sequence having at least 95% sequence identity to the VL sequence of SEQ ID NO: 16. In one aspect, the anti-Tn- MUC-1 antibody comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the VH sequence of SEQ ID NO: 12, and a VL comprising an amino acid sequence having at least 98% sequence identity to the VL sequence of SEQ ID NO: 16.

[0167] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence, and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising up to two (i.e. 0, 1 or 2) amino acid substitutions within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising one amino acid substitution within said amino acid sequence, and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising one amino acid substitution within said amino acid sequence. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 comprising two amino acid substitutions within said amino acid sequence, and a VL comprising the amino acid sequence of SEQ ID NO: 16 comprising two amino acid substitutions within said amino acid sequence. In one aspect, the substitutions are in the FR of the VH and VL.

[0168] In one aspect, the anti-Tn-MUC-1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 12, and a VL comprising the amino acid sequence of SEQ ID NO: 16. In one aspect, the anti-Tn-MUC-1 antibody comprises a VH and a VL comprising the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 16, respectively, including post- translational modifications of those sequences.

[0169] In one aspect, the anti-Tn-MUC-1 antibody comprises (a) a VH comprising an amino acid sequence having a single amino acid substitution in the VH sequence of SEQ ID NO: 12; and / or (b) a VL comprising an amino acid sequence having a single amino acid substitution in the VL sequence of SEQ ID NO: 16. In one aspect, the substitution is in the FR of the VH and / or VL. In one aspect, the anti-Tn-MUC-1 antibody comprises (a) a VH comprising an amino acid sequence having two amino acid substitutions in the VH sequence of SEQ ID NO: 12; and / or (b) a VL comprising an amino acid sequence having two amino acid substitutions in the VL sequence of SEQ ID NO: 16. In one aspect, the substitutions are in the FR of the VH and / or VL.

[0170] In one aspect, the anti-Tn-MUC-1 antibody of the invention comprises a human constant region. In one aspect, the anti-Tn-MUC-1 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 22 and 23 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 24 (human IgGi heavy chain constant domains CH1-CH2-CH3).

[0171] 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: 21. 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).

[0172] 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. 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.

[0173] / . Antibody Affinity

[0174] 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 < 250 nM, most particularly a KDof < 200 nM.

[0175] 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.

[0176] In a particular aspect, 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:

[0177] SPR is performed on a Biacore 8K instrument (Cytiva) at 25°C with PBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20 (Cytiva, Freiburg / Germany)) as running buffer.

[0178] Biotinylated Tn-MUC-1 peptide (Tn-glycosylated MUC-1 peptide Biotin-(O2Oc)2-RPAPG- S(GalNAc)-T(GalNAc)-APPAHGVT-amid, SEQ ID NO: 27) 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 four 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 1-8 (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 in an increasing concentration series of 0 - 50 - 100 - 200 - 400 - 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 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 from 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 was performed using Biacore Insight Software (Cytiva).

[0179] 2. Antibody Fragments

[0180] 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.

[0181] 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).

[0182] 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.

[0183] 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.

[0184] 3. Human Antibodies

[0185] In one aspect, the anti-Tn-MUC-1 antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0186] Human antibodies may be prepared by administering an immunogen to a transgenic animal (e.g. a mouse or a rabbit) that has been modified to produce full-length human antibodies or antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S. PatentNos. 6,075,181 and 6,150,584; U.S. PatentNo. 5,770,429; U.S. Patent No. 7,041,870, and U.S. Patent Application Publication No. US 2007 / 0061900). Human variable regions from full-length antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0187] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. See, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0188] Human antibodies may also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0189] 4. Library-Derived Antibodies

[0190] 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)).

[0191] 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.

[0192] 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).

[0193] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0194] 5. Multispecific Antibodies

[0195] In one aspect, the anti-Tn-MUC-1 antibody provided herein is a multispecific, e.g. a bispecific, antibody.

[0196] 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).

[0197] 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).

[0198] 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).

[0199] 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).

[0200] 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)).

[0201] 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)).

[0202] 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.

[0203] 6. Antibody Variants

[0204] 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

[0205] 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. Amino acids may be grouped according to common side-chain properties:

[0206] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0207] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu;

[0208] (4) basic: His, Lys, Arg;

[0209] (5) residues that influence chain orientation: Gly, Pro;

[0210] (6) aromatic: Trp, Tyr, Phe.

[0211] Conservative substitutions will entail exchanging members of one of these classes for another member in the class.

[0212] 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).

[0213] 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.

[0214] 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.

[0215] 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

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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).

[0220] 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.

[0221] 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

[0222] 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.

[0223] 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).

[0224] 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). 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.

[0225] 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).

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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. 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)).

[0231] 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.

[0232] 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.

[0233] 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.

[0234] For additional Fc region mutations see e.g. Abdeldaim and Schindowski, Pharmaceutics 15(10): 2402 (2023).

[0235] 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

[0236] 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.

[0237] 7. I minu noconjugates

[0238] 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.

[0239] 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).

[0240] 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.

[0241] 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.

[0242] In another aspect, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.

[0243] 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.

[0244] 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.

[0245] 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-(A- 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).

[0246] 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.

[0247] 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).

[0248] B. Recombinant Methods and Compositions

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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).

[0253] 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.

[0254] 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.

[0255] 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).

[0256] 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.

[0257] 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).

[0258] 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).

[0259] 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.

[0260] 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.

[0261] 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. C. Methods and Compositions for Diagnostics and Detection

[0262] 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.

[0263] 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.

[0264] 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.

[0265] D. Pharmaceutical Compositions

[0266] 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.

[0267] 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.

[0268] 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).

[0269] 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.

[0270] 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.

[0271] E. Therapeutic Methods and Routes of Administration

[0272] Any of the anti-Tn-MUC-1 antibodies provided herein may be used in therapeutic methods.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] F. Articles of Manufacture

[0284] 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

[0285] IV. EXAMPLES

[0286] 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.

[0287] Example 1 - General methods

[0288] Recombinant DNA Techniques

[0289] 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 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.

[0290] Gene Synthesis

[0291] 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.

[0292] Production of IgG and TCB antibodies

[0293] 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.

[0294] 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 2A.

[0295] The antibodies described herein were produced using deep well plates or shake flasks with batch or fed-batch 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 6-12 days after transfection.

[0296] Quantification of protein titer

[0297] 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.

[0298] Purification of IgG and TCB antibodies

[0299] 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.

[0300] Preparation of Fab fragments

[0301] IgG material was prepared for enzymatic cleavage by dialyzing it into a buffer solution consisting of 75 mM potassium phosphate (KP), 150 mM sodium chloride (NaCl), and 2 mM ethylenediaminetetraacetic acid (EDTA) at a pH of 7.2. Following dialysis, the IgG was subjected to cleavage with the enzyme papain. After the enzymatic cleavage, the mixture was processed through chromatography using MabSelect resin (MabSelect, Cytivia). Fab fragments were collected from the flow-through. Subsequently, the Fab-containing flow-through was further purified using size exclusion chromatography (Superdex 200, Cytiva).

[0302] Analytics of IgG and TCB antibodies

[0303] 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).

[0304] 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.

[0305] Cell engineering

[0306] 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 followed by single cell cloning. Successful homozygous 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 human embryonic kidney cell line 293 A and the breast cancer cell line MCF-7. The resulting cell lines expressing the Tn glycan were named HEK 293A COSMC KO and MCF7 COSMC KO, respectively.

[0307] In the same way, the double knockout cell lines HEK 293 A COSMC KO-MUC1 KO and MCF7 COSMC KO-MUC1 KO were generated by inactivating the MUC-1 gene in HEK 293 A COSMC KO or MCF7 COSMC KO cells, respectively.

[0308] Since 293 A 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 wildtype background. The resulting cell lines were named HEK 293A COSMC KO + MUC1 and HEK 293 A + MUC1. In addition, a cell line overexpressing cynomolgus MUC-1 fused to an intracellular GFP domain was generated in the COSMC KO background. This cell line was named HEK 293 A COSMC KO + cynoMUCl.

[0309] Tn glycans are naturally expressed by Jurkat and Jurkat-derived cell lines, such as the P329G- CAR-J reporter cells (Protein Eng Des Sei (2019) 32(5):207-218), due to the inactivation of the C1GALT1C1 gene. In order to remove this naturally occurring Tn glycosylation, which may interfere with the functional testing of anti-Tn-MUC-1 IgG, P329-CAR-J reporter cells were randomly transfected with an expression plasmid for human C1GALT1C1. Cells that had stably integrated the recombinant DNA were selected with G418. After single-cell cloning, the suppression of Tn glycosylation was proven by Tn-specific flow cytometry. The resulting cell line was named P329G-CARJ COSMC.

[0310] ELISA

[0311] Enzyme-linked immunosorbent assays (ELISA) were applied to evaluate the target binding of B-cell culture supernatants and recombinant antibody preparations at high throughput.

[0312] Biotinylated peptide or protein antigens were immobilized at a concentration of 8 nM in 25 pL PBS, 0.5% BSA, 0.05% Tween 20 on a streptavidin-coated 384-well microtiter plate (Microcoat / Thermofischer #11974998). Each of the following steps was followed by a threefold washing routine with 90 pL PBS: 1) incubation with primary antibody at increasing concentrations or at a single concentration for 1 h, 2) incubation with detection antibody (antirabbit IgG F(ab’)2 Donkey POD (Amersham #NA9340V, diluted 1 :3000) or anti-human IgG F(ab’)2 POD (Jackson #109-036-006, diluted 1 :6000)). 20-30 min after adding the substrate 3,3’, 5,5’ tetramethylbenzidine (TMB, Piercenet # 34021) the optical density was determined at 370 nm. The EC50 was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.

[0313] For a cell-based ELISA assay, cells were seeded in poly-D-lysine 384-well plates (Corning #354662) at a density of 105cells per well and incubated for 48 h at standard cell culture conditions. Following a wash step with 90 pL PBS, 0.1% Tween 20 (PBST), 10 pL primary antibody in PBS was added at a single or multiple concentrations and left for 2 hrs at 4°C. Unbound antibody was removed by wash step with 90 pL PBST. Bound antibodies were fixed by 15 min incubation at room temperature with 25 pL 0.05% glutaraldehyde (Sigma #G5882) in PBS. Excess glutaraldehyde was cleared using 3 wash steps with 90 pL PBST each. The antigen-antibody complex was detected using secondary antibodies (anti-rabbit F(ab’)2 Donkey POD, Amersham #A9340V, 1 : 1000) and the TMB substrate. The optical density was determined at 370 nm. If applicable the EC50 was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.

[0314] Surface plasmon resonance

[0315] Binding of humanized anti-Tn-MUC-1 immunoglobulins (Fab) to biotinylated Tn-MUC-1 peptides was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore 8K at 25°C with PBS-P as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20; Cytiva, Freiburg / Germany).

[0316] 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: 300 s, flow: 2 pl / min). Subsequently, biotinylated Tn-MUC-1 peptides were captured via streptavidin biotin coupling on flow channels 1-8 (contact time: 180 s, flow: 10 pl / min) with a concentration of 5 nM each. The anti-Tn-MUC-1 binders passed the surface in an increasing concentration series of 0 - 50 - 100 - 200 - 400 - 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). Flow cytometry

[0317] Flow cytometry was either performed with a BD FACSymphony or BD FACS Celesta flow cytometer.

[0318] For analysis with the BD FACSymphony device, target cells were harvested, washed once with FACS buffer (eBioscience™, #00-4222-57) 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.

[0319] 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) were added. After 20 minutes incubation at room temperature the cells were washed twice with PBS, resuspended in 100 pl FACS buffer per well and analyzed.

[0320] If the BD FACSCelesta was used, the procedure was as follows:

[0321] Target cells were harvested, counted and adjusted to a cell concentration of 1.25 x 106cells / ml in PBS. The cell suspension was dispensed to a 384 round-bottom well plate at a volume of 40 pl per well. Molecules were serially diluted with PBS at steps of 1 :5, starting at 150 nM in a 96 well plate. 10 pl per well was added to target expressing cells. The plates were incubated for 60 min at 4°C. The plates were centrifuged for 3 min at 300 x g, supernatant was removed and cells were washed with PBS. Afterwards, 25 pl of secondary antibody (A647 goat anti-human IgG (H+L), Invitrogen, #A21445 at a dilution of 1 : 100) was added. After 30 min of incubation for 30 min at 4°C in the dark, cells were washed twice with FACS buffer, resuspended in 30 pl FACS buffer and analyzed using the FACSCelesta (BD) instrument.

[0322] Activation of an anti-P329G CAR-Jurkat (CAR-J) reporter cell line

[0323] Jurkat NFAT-luciferase reporter cells express a luciferase reporter gene driven by an NFAT- response element. Activation of the Jurkat-NF AT -luciferase reporter cells by TCR signaling results in NFAT-mediated luminescence.

[0324] Jurkat-NF AT-luciferase reporter cells have been stably transduced to produce a chimeric antigen receptor (CAR) directed against the P329G mutation in the Fc-part of effector-silenced human IgGi-antibodies. These cells were named P329G-CAR-J cells and have been used as effector cells for the identification and characterization of IgGs prior to conversion into T cell bispecific antibodies (Protein Eng Des Sei (2019) 32(5):207-218). Stable recombinant expression of C1GALT1C1 in P329G-CAR-J cells yielded a cell line with non-detectable Tn glycosylation, which we named P329G-CARJ COSMC.

[0325] P329G-CAR-J COSMC cells were used as effector cells for the characterization of anti-Tn- MUC-1 IgGi with a P329G-mutated Fc. MCF-7 cells with inactivated C1GALT1C1 gene (MCF7 COSMC KO or MCF7 COSMC K0-MUC1 KO) and consequently increased Tn antigen expression were used as target cells. Human Bronchial Epithelial Cells (HBEpiC; ScienCell Research Laboratories, #3210) were used as normal human control cells lacking Tn glycosylation. The assay was performed as follows:

[0326] P329G-CAR-J COSMC cells were harvested and resuspended at a density of 3 x 106cells / ml in assay medium (RPMI 1640, Gibco #72400) containing 10% FBS. 30 pl of the cell suspension was transferred into white, flat-bottom, cell-culture treated 96-well plates. Target cells were detached using trypsin (Gibco), washed once with PBS and re-suspended at a density of 1 x 106cells / ml in the assay medium. 30 pl of the cell suspension was transferred to the plate containing the P329G-CAR-J COSMC cells. Antibodies were diluted in assay medium at indicated concentrations and 30 pl of molecules were added to the target cells. The plates were incubated for 24 h at 37°C and 5% CO2.

[0327] The ONE-Glo™ Luciferase Assay System was used to detect luciferase reporter gene expression in P329G-CAR-J COSMC cells. In brief, ONE-Glo™ substrate was adjusted to room temperature and 90 pl of the substrate was added to each well. After 15 minutes incubation at room temperature, luminescence was measured using a Perkin Elmer EnVision® 2104 instrument.

[0328] Primary T cell mediated tumor cell killing

[0329] The functional characterization of T cell bispecific antibodies (TCBs) was performed by assessing T cell mediated killing of tumor cells.

[0330] 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.

[0331] 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.

[0332] 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).

[0333] 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.

[0334] Example 2 - Generation of anti-Tn-MUC-1 antibodies by immunization

[0335] Immunization of wildtype and transgenic rabbits

[0336] For immunization, New Zealand White (NZW) rabbits obtained from Charles River Laboratories International, Inc. and Roche proprietary transgenic rabbits, expressing a humanized antibody repertoire were used. Transgenic rabbits comprising a human immunoglobulin locus are reported in WO 2000 / 46251, WO 2002 / 12437, WO 2005 / 007696, WO 2006 / 047367, US 2007 / 0033661, and WO 2008 / 027986 (all incorporated herein by reference in their entirety). The animals were housed according to the Appendix A “Guidelines for accommodation and care of animals” in an A AAL AC -accredited animal facility. All animal immunization protocols and experiments were approved by the Government of Upper Bavaria and performed according to the German Animal Welfare Act and the Directive 2010 / 63 of the European Parliament and Council.

[0337] NZW rabbits (n=2) or transgenic rabbits (n=3) expressing a humanized antibody repertoire were immunized with a Tn-MUC-1 40mer peptide coupled to keyhole limpet hemocyanin (Tn- 40mer-KLH; Table 1). NZW rabbits received 6 consecutive immunizations at weeks 0, 1, 4, 9, 13 and 17. Transgenic rabbits received 6 consecutive immunizations at weeks 0, 1, 2, 7, 10 and 14. 200 - 400pg of the immunogen formulated with either complete Freund’s adjuvant or a combination of TLR agonists were injected, while intradermal, intramuscular and subcutaneous administration routes were alternated during the immunization procedure.

[0338] Blood (max. 10% of estimated total blood volume) was retrieved at days 6 to 8 post immunizations, starting from the third immunization onwards. Serum was prepared, which was used for antigen-specific titer determination by ELISA, and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the B cell cloning process.

[0339] Determination of serum titers

[0340] 96-well plates were coated with Tn-40mer, corel-40mer, non-glyco-40mer, Tn-MUC5AC- 14mer or KLH (Table 1) at 3 pg / mL, 100 pL / well, in PBS, followed by: blocking of the plate with 2 % Crotein C in PBS, 200 pL / well; application of serial dilutions of antisera, in duplicates, in 0.5 % Crotein C in PBS, 100 pL / well; detection with (1) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch / Dianova #711-036-152; 1 / 16 000) for all rabbit sera, (2) biotinylated goat anti-human kappa antibody (Southern Biotech / Biozol #2063-08, 1 / 5 000) and streptavidin-HRP for sera from transgenic rabbits only; diluted in 0.5 % Crotein C in PBS, 100 pL / well. For all steps, plates were incubated for 1 h at 37°C. Between all steps, plates were washed 3 times with 0.05% Tween 20 in PBS. Signal was developed by addition of BM Blue POD Substrate soluble (Roche), 100 pL / well; and stopped by addition of 1 M HC1, 100 pL / well. Absorbance was read out at 450 nm, against 690 nm as reference. Titer was defined as dilution of antisera resulting in half-maximal signal.

[0341] B-cell cloning and screening

[0342] Isolation of rabbit peripheral blood mononuclear cells (PBMC)

[0343] From every immunized rabbit (NZW or transgenic) 4 blood samples were taken during the course of the immunization procedure and fed into the B cell cloning process. EDTA containing whole blood was diluted twofold with lx PBS (PAA, Pasching, Austria) before density centrifugation using lympholyte mammal (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the specifications of the manufacturer. The PBMCs were washed twice with lx PBS.

[0344] EL-4 B5 medium

[0345] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone,

[0346] Logan, UT, USA), 2 mM glutamine, 1% penicillin / streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM P-mercaptoethanol (Gibco, Paisley, Scotland). of cells

[0347] 6-well plates coated with KLH were used to deplete KLH-specific B-cells as well as macrophages / monocytes and non-specific B-lymphocytes through nonspecific adhesion. Each well was filled at maximum with 4 mL medium and up to 6 x 106PBMCs from the immunized rabbit and allowed to bind for 1 h at 37°C in the incubator. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step or directly subjected to B-cell sorting via flow cytometry. enrichment of B-cells

[0348] 6-well plates covered with either a monolayer of HEK 293 A COSMC KO cells or Tn-40mer peptide were seeded with up to 6 x 106PBLs per 4 mL medium, which were allowed to bind for 1 h at 37°C in the incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with lx PBS. The remaining sticky cells were detached by trypsin for 10 min. at 37°C in the incubator. Trypsinization was stopped with EL-4 B5 medium. The cells were kept on ice until the immune fluorescence staining.

[0349] Immunofluorescence staining and Flow Cytometry

[0350] The anti-rabbit IgG FITC antibody (Abeam, Cambridge, United Kingdom) and the anti-human Ig, K light chain APC antibody (BD Biosciences, New Jersey, USA) were used for single cell sorting. For surface staining, cells from the preceding depletion and enrichment steps were incubated with the aforementioned antibodies in PBS and incubated for 45 min in the dark at 4°C. After staining, the PBMCs were washed twice with ice cold PBS. Finally the PBMCs were resuspended in ice cold PBS and immediately subjected to FACS analysis and sorting. Propidium iodide at a concentration of 5 pg / mL (BD Pharmingen, San Diego, CA, USA) was added prior to the FACS analysis to discriminate between dead and live cells. Cells from transgenic rabbits with a double positive signal (rabbit IgG, human Ig K light chain) and cells from wildtype rabbits with a single positive signal (rabbit IgG) were sorted into 96-well plates at a density of one cell per well. A Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences, USA) were used for single cell sorting.

[0351] B-cell cultivation Single sorted rabbit B-cells were incubated in 96-well plates with 200 pL / well EL-4 B5 medium containing Pansorbin Cells (1 :100000) (Calbiochem (Merck), Darmstadt, Germany), 0,35 ng / ml phorbol-12-myristat-13-acetat (PMA, Sigma-Aldrich, St. Louis, USA), a mouse cytokine mix consisting of TNF, IL- lb, IL-2, IL-10 and IL-6 (Miltenyi Biotec, Bergisch Gladbach, Germany) and gamma-irradiated murine EL-4-B5 thymoma cells (2.0 x 104 / well) for 7 days at 37°C in an atmosphere of 5% CO2 in the incubator. The supernatants of the B-cell cultivation were harvested for screening and the remaining cells were immediately frozen at -80°C in 100 pL RLT buffer (Qiagen, Hilden, Germany) for recovering the V region by PCR.

[0352] Screening of B-cell supernatants

[0353] Supernatants from B-cells were screened by ELISA for Tn-MUC-1 specific clones using a panel of antigens: Tn-40mers, which are Tn-glycosylated dimers of the MUC-1 standard repeat, and HEK 293A COSMC KO cells, which express Tn glycans and low levels of MUC-1, were used as target antigens for positive screening. In addition the following control antigens were used for negative screening: Corel-40mers (surrogate antigen for MUC-1 with normal O- glycosylation), non-glyco-40mers (surrogate antigen for non-glycosylated MUC-1), Tn- MUC5AC-14mer (non-related Tn-glycosylated antigen, to exclude mere Tn-glycan binders), KLH ( antigen carrier protein used for immunization against Tn-MUC-1 40mer) and wildtype HEK 293 A cells (for an overview of screening antigens and the desired binding properties see Table 1)

[0354] All B-cell colonies showing binding to either Tn-40mer peptide or HEK 293A COSMC KO cells or both, but no binding to any of the control antigens, were selected and subjected to B- cell PCR.

[0355] Table 1. Antigens for immunization and screening.

[0356] B-cell PCR and recombinant expression of antibodies from PCR pools

[0357] PCR amplification of V-domains

[0358] Total RNA was prepared from B-cell lysates (resuspended in RLT buffer - Qiagen, #79216) using the NucleoSpin 96 RNA kit (Macher ey&Nagel, #740709.4) according to manufacturer’s protocol. RNA was eluted with 60 pL RNAse free water. 6 pL of RNA was used to generate cDNA by reverse transcriptase reaction using the Superscript III First-Strand Synthesis SuperMix (Invitrogen, #18080400) and an oligo-dT-primer according to the manufacturer’s instructions. All steps were performed on a Hamilton ML Star System. 4 pL of cDNA were used to amplify the immunoglobulin heavy and light chain variable regions (VH and VL), respectively, with the AccuPrime Pfx Supermix (Invitrogen, #12344040) in a final volume of 50 pL. Suitable primers were prepared by metabion GmbH (Planegg-Martinsried, Germany). All forward primers (wildtype and transgenic) were specific for the signal peptide of VH and VL, respectively. Reverse primers for wildtype rabbits were specific for the J gene segments of VH and VL, respectively. Reverse primers for transgenic rabbits were specific for the J gene segments of the heavy chain and specific for the constant region of the light chain.

[0359] 8 pL of 50 pL PCR solution was loaded on a 48 E-Gel 2% (Invitrogen, #G800802). Positive PCR reactions for both chains were purified using the NucleoSpin 96 PCR Clean-up kit (Macher ey&Nagel, #740658.4) according to manufacturer’s protocol and eluted with 75 pL elution buffer. All purification steps were performed on a Hamilton ML Starlet system.

[0360] Molecular cloning of PCR

[0361] For recombinant expression of rabbit monoclonal bivalent antibodies, PCR-products coding for VH or VL were cloned into expression vectors by the overhang cloning method (Biotechniques. 1992 Oct;13(4):515-8;Nat Methods. 2007 Mar;4(3):251-6). T Two variants of the basic expression plasmid were used: one plasmid containing the human IgG constant region (with P329G L234A L325A (“PGLALA”) mutation) designed to accept the VH regions and one plasmid containing human kappa LC constant region to accept the VL regions. Linearized expression plasmids coding for the kappa or gamma constant region and VL / VH inserts were amplified by PCR using overlapping primers. Purified PCR products were incubated with T4 DNA-polymerase which generated single-strand overhangs. The reaction was stopped by dCTP addition. In the next step, plasmid and insert were combined and incubated with recA which induced site specific recombination. The recombinant plasmids were transformed into E.coli (Invitrogen, #C409610). The next day, liquid overnight cultures were purified by NucleoSpin 96 Plasmid kit (Machery&Nagel, #740625.4) according to manufacturer’s protocol. All liquid handling steps of cloning, transformation and plasmid preparation were performed on an Eppendorf epMotion 5075. An aliquot was used for Sanger DNA sequencing of VH and VL regions (SequiServe GmbH, Vatterstetten, Germany).

[0362] Recombinant expression of antibodies from PCR pools

[0363] Recombinant antibodies were transiently expressed in Expi293 cells (human embryonic kidney cell line 293 -derived), which were cultivated in Expi293 Expression Medium (Gibco, #A1435101). For transfection, the ExpiFectamine 293 Transfection kit (Gibco, #A14524) was used. Antibodies were expressed from the non-clonal expression plasmids described above. Transfections were performed as specified in the manufacturer’s instructions. Recombinant protein-containing cell culture supernatants were harvested six days after transfection. Supernatants were stored at reduced temperature (e.g. -80°C) until purification or purified the next day.

[0364] Example 3 - Identification of Tn-MUC-1 specific antibodies

[0365] Primary Screening of recombinant antibodies

[0366] ELISA screening

[0367] Similar to the supernatants from B-cells, recombinant antibodies from transient expression of non-clonal plasmid DNA in Expi293 cells were screened by ELISA for Tn-MUC-1 specific binders. Again, the Tn-40mers and HEK 293 A CO SMC KO cells were used as target antigens for positive screening whereas corel-40mers, non-glyco-40mers, Tn-MUC5AC-14mer and KLH were used as control antigens for counter screening (Table 1).

[0368] Table 1 provides an overview of antigens and the desired binding property. All antibodies showing binding to either Tn-40mer peptide or HEK 293 A COSMC KO cells or both, but no binding to any of the control antigens, were selected.

[0369] For a more sensitive detection of antibodies that show non-specific binding, especially to normally glycosylated MUC-1, the antibodies were tested by flow cytometry with normal HEK 293 A cells as well as HEK 293 A cells overexpressing a human MUC-1 -GFP fusion protein (HEK 293 A +MUC1). All antibodies that showed a signal above the isotype control with either cell line were deselected.

[0370] Flow cytometric screening

[0371] In order to differentiate between binders that are highly specific for Tn-glycosylated MUC-1 and others that have a broader specificity for Tn-glycosylated antigens, HEK 293A COSMC KO +MUC1 cells and the corresponding HEK 293 A COSMC KO cells with inactivated MUC1 genes (HEK 293 A COSMC K0-MUC1 KO) were used in an additional flow cytometry experiment. According to the relative signal intensities of both assays the binders were assigned to three groups: 1) highly Tn-MUC-1 specific (MFI with MUC1 KO cells at the level of the isotype control), 2) strong preference for Tn-MUC-1 (MFI with MUC1 KO cells strongly reduced compared to MUC-1 positive cell) and 3) broadly Tn-specific (equally high MFI with MUC-1 overexpressing cells and MUC1 KO cells). The amino acid sequence of all binders that had passed the primary screening was determined by DNA sequencing and back translation. Redundant sequences were eliminated. A representative plasmid DNA clone was isolated for each unique binder and used for recombinant expression and supply of the secondary screening.

[0372] Secondary screening

[0373] ELISA-based epitope binning and assessment of cross-reactivity with cynomolgus monkey Tn- MUC-1

[0374] In order to further differentiate between binders of various properties, we divided the 40meric human Tn-MUC-1 repeat antigen into three overlapping Tn-glycopeptides: hu Tn-Binl, hu Tn- Bin2 and hu Tn-Bin3 and tested the antibodies for their specificity towards these epitopes by ELISA. In addition, we generated the homologous Tn-peptides cyno Tn-Binl, cyno Tn-Bn2 and cyno Tn-Bin3 based on the cynomolgus MUC-1 repeat sequence in order to test the binders’ cross reactivity. For an overview of these peptides see Table 2. The synthesis of the cynomolgus Tn-40mer repeatedly failed so that it was not available for testing of crossreactivity. The human Tn-40mer and the non-glyco-40mer were used as positive and negative control respectively. For 24 antibodies considerable binding to any of the cynomolgus Tn- MUC-1 peptides was detected. Table 3 shows the EC50 values of their binding to Tn-40mer together with their epitope binning properties. None of the antibodies bound to the nonglycosylated control peptide.

[0375] Table 2. Overview of peptides for epitope binning.

[0376] Example 3 - Functional characterization of cynomolgus-cross reactive anti-Tn-MUC-1 antibodies

[0377] The IgGs of Table 3 where further characterized by assessing their ability to activate P329G- CARJ COSMC reporter cells, co-cultured with either MCF7 COSMC KO, MCF7 COSMC KO-

[0378] MUC1 KO or HBEpiC cells to test for Tn-MUC-1 specificity. HBEpiC cells were used as control cells lacking Tn glycosylation. Figure 2 shows the dose-dependent activity of the molecules in comparison with GO2 IgG (P1AA9754, SEQ ID NOs 17 (VH) and 18 (VL); see also see WO 2019 / 083506). All tested IgGs showed no or little activity with HBEpiC off-target cells. Whilst the inactivation of the MUC1 gene in target cells had little impact on the activity of GO2 IgG, reporter cell activation of almost all new IgGs was strongly reduced in COSMC K0-MUC1 KO double knockout cells. Molecules with a similar or better potency according to their EC50 value and higher selectivity for Tn-MUC-1 compared to GO2 were selected and converted to TCBs (Table 4).

[0379] Table 4. EC50 values of CAR-J assay.

[0380] * human

[0381] The TCBs of Table 4 were tested for T-cell mediated tumor cell killing. Figure 3 depicts results from a T cell mediated tumor cell killing assay performed with MCF7 COSMC KO as target cells and PBMCs as effector cells. Potential off-target activity was again tested with HBEpiC cells. P1AD9478, a non-target binding TCB, was used as a negative control. All selected molecules, except a single one, showed lower EC50 values than the GO2 TCB (P1AD8341). Corresponding EC50 values and top plateaus are listed in Table 5. All TCBs showed no or very little activity against HBEpiC cells. P1AG5361 and P1AG5364 were selected for further characterization due to their fully human nature and higher top plateau compared to the other two fully human TCBs Pl AG5360 and Pl AG5363. In addition, Pl AG5363 also showed a high EC 50 value.

[0382] Table 5. EC50 values of killing assay.

[0383] In order to test P1AG5361 and P1AG5364 for their Tn-MUC-1 specificity we performed another T cell mediated tumor cell killing assay using MCF7 COSMC KO or MCF7 COSMC K0-MUC1 KO as target cells (Figure 4). TCBs of known anti-Tn-MUC-1 binders GO2 (P1AD8341, SEQ ID NOs 17 (VH) and 18 (VL); see also WO 2019 / 083506) and 4AG (P1AE4887, SEQ ID NOs 19 (VH) and 20 (VL); see also WO 2020 / 006449) were used as controls. The GO2 TCB was active against MCF7 COSMC KO as well as MCF7 COSMC KO- MUC1 KO cell, indicating that the activity of the GO2 TCB was not impacted by the inactivation of the MUC1 gene. This suggests that the GO2 TCB can effectively bind to Tn antigens other than Tn-MUC-1. The 4AG TCB was poorly active against MCF7 COSMC KO cells indicated by the low upper plateau. Furthermore, the activity of 4AG TCB was completely abolished by the inactivation of MUC-1 expression. In contrast, P1AG5361 and P1AG5364 were highly active against MCF7 COSMC cells but much less active against MCF7 COSMC KO-MUC1 KO. This suggests that P1AG5361 and P1AG5364 combine high T-cell mediated killing activity with high selectivity towards Tn-MUC-1 compared to other Tn-modified antigens. EC50 and upper plateau values are listed in Table 6.

[0384] Table 6. Killing assay with selected TCBs.

[0385] * not calculable / top plateau not reached The specificity of the Tn-MUC-1 binding moieties of TCBs P1A5G5361 and P1AG5364 was further evaluated by FACS using the corresponding IgGs P1AG0639 and P1AG0648, respectively. A panel of HEK 293 A-derived cell lines was probed with a serial dilution of these antibodies and analyzed by flow cytometry. The following cell lines were used:

[0386] • HEK 293 A: wild-type cells

[0387] • HEK 293 A + MUC1 : HEK 293 A cells that overexpress human MUC-1 fused to an intracellular GFP domain

[0388] • HEK 293A COSMC KO: HEK 293A cells engineered to express the Tn O-glycan by knock-out of the C1GALT1C1 (COSMC) gene

[0389] . HEK 293 A COSMC KO + MUC1 : HEK 293 A COSMC KO cells that overexpress human MUC1 fused to an intracellular GFP

[0390] . HEK 293A COSMC K0-MUC1 KO: HEK 293A COSMC KO cells with additional knock-out of the MUC1 gene

[0391] Figure 5 shows dose-dependent binding of P1AG0639 and P1AG0648 in comparison to GO2 IgG (P1AA9754). With P1AG0639 and P1AG0648, substantial binding was only observed to HEK 293 A COSMC KO + MUCE This further demonstrates the high specificity of P1AG0639 and P1AG0648 towards Tn-glycosylated MUC-1 compared to normal MUC-1 or other Tn- glycosylated antigens. In contrast, GO2 IgG bound to all cell lines with inactivated COSMC gene, independent of whether MUC-1 was overexpressed, knocked-out or kept untouched.

[0392] Cross-reactivity of TCBs P1A5G5361 and P1AG5364 towards cynomolgus Tn-MUC-1 was determined by flow cytometry using engineered HEK 293A COSMC KO cells that either overexpress cynomolgus or human MUC-1 fused to intracellular GFP. Figure 6 shows dose dependent binding of P1A5G5361 and P1AG5364 towards either cell line. Corresponding EC50 values were determined and are listed in Table 7. Both TCBs showed significant binding to cells overexpressing either human of cyno Tn-MUC-1 with similar EC50 values. As shown before, both binders don’t bind to HEK 293A COSMC KO without MUC-1 overexpression or non-gly coengineered HEK293A cells overexpressing MUC-1 (see Figure 5). Consequently, the binding of P1A5G5361 and P1AG5364 is truly dependent on human or cynomolgus Tn- MUC-1 overexpression and demonstrates species cross-reactivity.

[0393] Table 7. Cyno cross-reactivity by FACS.

[0394] The monovalent affinity of IgG Pl AG0639 was determined by SPR using the corresponding Fab fragment as analyte and biotinylated Tn-MUC-1 peptides as ligands. Two different biotinylated ligands were used, first Tn-40mer representing two tandem copies of fully Tn- glycosylated MUC-1 repeats (Table 1) or hu Tn-Bin3 (Table 2) representing a Tn-glycosylatd fragment of the MUC-1 repeat. Similar KD values of 1.68E-7 and 2.33E-7, respectively, were determined for both antigens confirming the previous ELISA-based epitope binning of P1AG0639.

[0395] able 3. Epitope binning by ELISA.

[0396]

[0397]

[0398] * * *

[0399] 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

CLAIMS1. An antibody that binds to Tn-MUC-1, wherein the antibody comprises(i) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; or(ii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

2. The antibody of claim 1, wherein(i) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VH sequence of SEQ ID NO: 4; and / or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VL sequence of SEQ ID NO: 8; or(ii) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VH sequence of SEQ ID NO: 12; and / or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VL sequence of SEQ ID NO: 16.

3. The antibody of claim 1 or 2, wherein(i) the VH comprises the amino acid sequence of SEQ ID NO: 4 and the VL comprises the amino acid sequence of SEQ ID NO: 8; or(ii) the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO: 16.

4. An antibody that binds to Tn-MUC-1 comprising(i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8; or(ii) a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16.

5. The antibody of any one of claims 1 to 4, 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.

6. The antibody of any one of claims 1 to 4, wherein the antibody comprises an Fc region, particularly an IgGFc region, more particularly an IgGi Fc region, most particularly a human IgGi Fc region.

7. The antibody of any one of claims 1 to 4, 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.

8. The antibody of any one of claims 1 to 7, wherein the antibody is a multispecific antibody, particularly a bispecific antibody, optionally wherein the multispecific antibody binds to Tn-MUC-1 and to CD3.

9. Isolated polynucleotide encoding the antibody of any one of claims 1 to 8.

10. A host cell comprising the polynucleotide of claim 9.

11. A method of producing an antibody that binds to Tn-MUC- 1 , comprising culturing the host cell of claim 10 under conditions suitable for the expression of the antibody, and optionally further comprising recovering the antibody from the host cell.

12. An antibody that binds to Tn-MUC-1 produced by the method of claim 11.

13. A pharmaceutical composition comprising the antibody of any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

14. The antibody of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 for use as a medicament.

15. The antibody of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 for use in the treatment of cancer.

16. Use of the antibody of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 in the manufacture of a medicament.

17. Use of the antibody of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 in the manufacture of a medicament for the treatment of cancer.

18. 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 8 or the pharmaceutical composition of claim 13.

19. The invention as described hereinbefore.

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