Bispecific antibody constructs directed against MUC17 and CD3

Abstract

To provide bispecific antibody construct directed to MUC17 and CD3.SOLUTION: The present invention provides bispecific antibody constructs characterized by comprising a first domain binding to MUC17, a second domain binding to an extracellular epitope of the human and Macaca CD3ε chain, and optionally a third domain which is a specific Fc modality. Moreover, the invention provides a polynucleotide, encoding the antibody construct, a vector comprising this polynucleotide, host cells expressing the construct, and a pharmaceutical composition comprising the same.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to biotechnology products and methods, in particular to bispecific antibody constructs, their formulation and their uses. [Background technology]

[0002] Among the most rapidly and promisingly developed therapeutics are protein-based drugs, which already play an important role in almost every medical field and are among the fastest growing therapeutics in (pre)clinical development and on the market (Leader, Nature Reviews Drug Discovery 2008 Jan 7, 21-39). Compared with small molecule chemical drugs, protein drugs have high specificity and activity at relatively low concentrations, and usually provide treatment for diseases with high impact, such as various cancers, autoimmune diseases and metabolic disorders (Roberts, Trends Biotechnol. 2014 Jul; 32(7): 372-80; Wang, Int J Pharm. 1999 Aug 20; 185(2): 129-88).

[0003] Such novel protein-based pharmaceuticals include, for example, bispecific (monoclonal) antibodies, which are usually capable of binding simultaneously to two different types of antigens. They are known in several structural formats and are currently being explored for applications in cancer immunotherapy and drug delivery (Fan, Gaowei; Wang, Zujian; Hao, Mingju; Li, Jinming (2015). "Bispecific antibodies and their applications". Journal of Hematology & Oncology. 8:130).

[0004] Bispecific antibodies can be IgG-like, i.e., full-length bispecific antibodies, or non-IgG-like bispecific antibodies that are not full-length antibody constructs. Full-length bispecific antibodies usually retain the normal monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab sites bind different antigens. Non-full-length bispecific antibodies may completely lack the Fc region. These include chemically linked Fabs consisting of only the Fab region, as well as various types of bivalent and trivalent single-chain variable fragments (scFv). There are also fusion proteins that mimic the variable domains of two antibodies. An example of such a format is the Bispecific T Cell Engager (BiTE®) (Yang, Fa; Wen, Weihong; Qin, Weijun (2016). "Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies". International Journal of Molecular Sciences. 18(1):48).

[0005] Bispecific antibody-derived molecules such as BiTE® antibody constructs are recombinant protein constructs composed of two flexibly linked antibody-derived binding domains. One binding domain of the BiTE® antibody construct is specific for a selected tumor-associated surface antigen on target cells, and the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. Due to their special design, BiTE® antibody constructs are uniquely suited to transiently link T cells to target cells and simultaneously potently activate the intrinsic cytolytic capacity of T cells against target cells. Significant further developments of the first generation BiTE® antibody constructs deployed in the clinic as AMG103 and AMG110 (see WO 99 / 54440 and WO 2005 / 040220) have led to the development of novel antibody constructs that can be used to target T cells. provided a bispecific antibody construct that binds to a context-independent epitope at the N-terminus of the CD3ε chain (WO 2008 / 119567). The BiTE® antibody construct that binds to this selected epitope not only does not show cross-species specificity for human and Macaca or Callithrix jacchus, Saguinus oedipus or Saimiri sciureus CD3ε chains, but also, because it recognizes this specific epitope (instead of the epitope of the CD3-binding molecule previously described in the bispecific T cell engaging molecule), does not show non-specific activation of T cells to the same extent as observed with previous generation T cell engaging antibodies. This reduced T cell activation is associated with less or reduced T cell redistribution in patients, the latter of which was determined to be a risk of side effects, for example with pasotuximab.

[0006] The antibody constructs described in WO 2008 / 119567 are characterized by rapid clearance from the body; therefore, although they can reach most parts of the body quickly, their in vivo application may be limited by their short persistence in vivo. On the other hand, their concentration in the body can be readily adapted and fine-tuned. Due to the short in vivo half-life of these small single-chain molecules, continuous administration by continuous intravenous infusion is used to achieve a therapeutic effect. However, new bispecific antibody constructs are available that have more favorable pharmacokinetic properties, including longer half-lives. A long half-life is generally useful in in vivo applications of immunoglobulins, particularly antibodies, and most particularly antibody fragments or constructs of small size, for example for patient compliance.

[0007] Mucins have been identified as interesting markers of inflammatory and cancerous diseases. Mucins are high molecular weight glycoproteins characterized by high levels of O-glycosylation at serine and threonine residues within tandem repeat domains (Johansson and Hansson, Nat. Rev. Immunology 2016). There are at least 20 mucin family members, including secreted and transmembrane proteins, expressed by epithelial cells in various tissues (Corfield, Biochim. Biophys. Acta 2013). The primary function of mucins is in the structure and regulation of the mucosal layer that forms a protective barrier between epithelial cells and the environment (Hollingsworth and Swanson, Nat. Rev. Cancer 2004; Hattrup and Gendler, Annu. Rev. Physiol. 2008). Transmembrane mucins also play a role in cellular signaling, including regulation of proliferation and apoptosis, and tumorigenesis (Hollingsworth and Swanson, Nat. Rev. Cancer 2004). Among the mucins, mucin 17 (MUC17) is the first transmembrane mucin identified by its homology to MUC3 (Gum et al., Biochem. Biophys. Res. Comm. 2002).

[0008] Analysis of the complete coding sequence of MUC17 revealed that it has a central region of 61 tandem repeats, a large extracellular domain composed of an epidermal growth factor (EGF) domain, a sea urchin sperm protein, enterokinase and agrin (SEA) domain, and a second EGF domain. The SEA domain contains a putative cleavage site that is conserved in other mucins (Moniaux et al., J. Biol. Chem. 2006). MUC17 is a single-pass transmembrane protein with an 80 amino acid cytoplasmic tail that is intracellular (Moniaux et al., J. Biol. Chem. 2006). Expression of MUC17 in healthy adults is restricted to the apical surface of enterocytes lining the intestinal tract or mature absorptive epithelial cells (Moniaux et al., J. Biol. Chem. 2006; Johanasson and Hansson, Nat. Rev. Immune System. 2007). MUC17 is also expressed by the stomach and pancreas (Moniaux et al., J. Biol. Chem. 2006; Moehle et al., J. Mol. Med. 2006). The biological function of MUC17 is thought to be the maintenance of the integrity of the intestinal mucosal barrier, including through mucosal repair (Luu et al., Int. J. Biochem. Cell Biol. 2010; Resta-Lenert et al., Am. J. Physiology 2011; Johanasson and Hansson, Nat. Rev. Immunology 2016).

[0009] MUC17 is aberrantly expressed in some cancers. MUC17 mRNA was shown to be expressed in one pancreatic cancer cell line and three colon cancer lines (Gum et al. 2002). Immunohistochemistry confirmed the expression of MUC17 protein in pancreatic cancer (Moniaux et al. 2006). However, in colon cancer, MUC17 protein expression was shown to be reduced (Senapati et al. 2007). (e.g., J. Clin. Pathol. 2010). Nevertheless, the expression pattern of MUC17 makes it a potential target for the treatment of various forms of malignancies. Summary of the Invention [Problem to be solved by the invention]

[0010] Considering the conflicting meanings in the literature regarding MUC17 as a potential target for which pathologies, it is an object of the present invention to provide a bispecific antibody construct, such as a T cell engaging molecule, that clearly identifies a specific condition associated with upregulation of MUC17 and is particularly suitable for binding MUC17 in MUC17-associated conditions, preferably for use in the treatment of said specific conditions. Accordingly, the present invention provides an antibody construct characterized by comprising a first domain that binds MUC17, a second domain that binds to an extracellular epitope of human and non-human, e.g. macaque CD3 epsilon chain, and a third domain, preferably in a specific Fc format. Furthermore, the present invention provides a polynucleotide encoding the antibody construct, a vector comprising this polynucleotide and a host cell expressing the construct, as well as a pharmaceutical composition comprising the same. [Means for solving the problem]

[0011] In a first aspect, the present invention relates to a first domain that binds to MUC17, and A second domain that binds to an extracellular epitope of the human and macaque CD3ε chain It is envisaged that the present invention provides an antibody construct comprising:

[0012] In said aspect, it is further envisaged in the context of the present invention that the antibody construct comprises a third domain comprising two polypeptide monomers each comprising a hinge, a CH2 domain and a CH3 domain, said two polypeptide monomers being fused to each other via a peptide linker.

[0013] In said aspect, it is further envisaged in the context of the present invention to provide an antibody construct which is a single chain antibody construct. In said aspect, it is also envisaged in the context of the present invention to provide an antibody construct, wherein said third domain comprises, in amino to carboxy order: hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0014] In the above aspect, it is further envisaged in relation to the present invention to provide an antibody construct, wherein each of the polypeptide monomers has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 24.

[0015] In the above aspect, it is also envisaged in relation to the present invention to provide an antibody construct, wherein each of the polypeptide monomers has an amino acid sequence selected from SEQ ID NOs: 17-24.

[0016] In said aspect, it is further envisaged in the context of the present invention to provide an antibody construct, wherein the CH2 domain comprises an intradomain cysteine ​​disulfide bridge. In the above aspect, in relation to the present invention, (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) It is also envisaged to provide an antibody construct, wherein the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain.

[0017] In said aspect, it is also envisaged in the context of the present invention to provide an antibody construct, wherein the first domain and the second domain are fused to the third domain via a peptide linker.

[0018] In said aspect, it is relevant to the present invention that the antibody construct comprises, in order from amino to carboxyl: (a) the first domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) Secondary Domain It is also envisaged to provide an antibody construct comprising:

[0019] In the above aspect, in relation to the present invention, the antibody construct further comprises, in addition to (a) to (c), the following, in order from amino to carboxyl: (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12; (e) a first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (g) a second polypeptide monomer of the third domain. It is also envisaged to provide an antibody construct comprising:

[0020] In said aspect, it is further envisaged in the context of the present invention to provide an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO:528 (aa 4171-4296 according to uniprot Q685J3 numbering).

[0021] In said aspect, it is also envisaged in the context of the present invention to provide an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO:529 (aa 4184-4291 according to uniprot Q685J3 numbering).

[0022] In said embodiment, in the context of the present invention, the first domain of the antibody construct is It is further contemplated to provide an antibody construct that binds to an epitope within MUC17 corresponding to 530 (aa 4131-4243 according to uniprot Q685J3 numbering).

[0023] In said aspect, it is also envisaged in the context of the present invention to provide an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO:531 (aa 4244-4389 according to uniprot Q685J3 numbering).

[0024] In said aspect, it is further envisaged in the context of the invention to provide an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO:530 (aa 4131-4243 according to uniprot Q685J3 numbering) but does not bind to an epitope in MUC17 corresponding to SEQ ID NO:531 (aa 4244-4389 according to uniprot Q685J3 numbering).

[0025] In said aspect, it is also envisaged in the context of the invention to provide an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO:532 (aas 4171-4390 according to uniprot Q685J3 numbering) or SEQ ID NO:533 (aas 4184-4390 according to uniprot Q685J3 numbering) but does not bind to an epitope in MUC17 corresponding to SEQ ID NO:534 (aas 4291-4390 according to uniprot Q685J3 numbering) or an epitope in MUC17 corresponding to SEQ ID NO:535 (aas 4341-4390 according to uniprot Q685J3 numbering).

[0026] In said aspect, it is further envisaged in the context of the present invention to provide an antibody construct, wherein the VH VL configuration is characterized as 4 lambda 3. Nomenclature is known in the art.

[0027] In said embodiment, in the context of the present invention, the ratio between cytotoxicity and binding affinity (EC 50 / K D It is further envisaged to provide an antibody construct having an EC activity of 0.01 to 0.100, wherein the EC activity is expressed in pM and determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and the binding affinity is expressed in nM and determined by a surface plasmon resonance (SPR) assay, such as a Biacore assay. 50 Value and K D A factor of 1000 is introduced to improve readability, taking into account the dimensional difference between the values.

[0028] In said embodiment, in the context of the present invention, the ratio between cytotoxicity and binding affinity (EC 50 / K D )*1000 is less than 125, cytotoxicity is given in pM and is determined, for example, in NUGC-4 cells as target cells and huPBMCs as effector cells, and binding affinity is given in nM and is determined, for example, by a surface plasmon resonance based assay.

[0029] In said embodiment, in the context of the present invention, the ratio between cytotoxicity and binding affinity (EC 50 / K D It is further envisaged to provide an antibody construct, wherein cytotoxicity (EC)*1000 is less than 21, cytotoxicity is given in pM and is determined, for example, in NUGC-4 cells as target cells and huPBMCs as effector cells, and binding affinity is given in nM and is determined by a surface plasmon resonance based assay. Preferably, cytotoxicity (EC)*1000 is less than 21, cytotoxicity is given in pM and is determined, for example, in NUGC-4 cells as target cells and huPBMCs as effector cells, and binding affinity is given in nM and is determined by a surface plasmon resonance based assay. 50 ) is <100 pM, and the binding affinity (K D ) is <25nM.

[0030] In said aspect, in the context of the present invention, the first binding domain comprises (a) CDR-H1 as set forth in SEQ ID NO: 33, CDR-H2 as set forth in SEQ ID NO: 34, and CDR-H3 as set forth in SEQ ID NO: 35; (b) CDR-H1 as set forth in SEQ ID NO: 44, CDR-H2 as set forth in SEQ ID NO: 45, and CDR-H3 as set forth in SEQ ID NO: 46; (c) CDR-H1 as set forth in SEQ ID NO:55, CDR-H2 as set forth in SEQ ID NO:56, and CDR-H3 as set forth in SEQ ID NO:57; (d) CDR-H1 as set forth in SEQ ID NO:66, CDR-H2 as set forth in SEQ ID NO:67, and CDR-H3 as set forth in SEQ ID NO:68; (e) CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 78, and CDR-H3 as set forth in SEQ ID NO: 79; (f) CDR-H1 as set forth in SEQ ID NO: 88, CDR-H2 as set forth in SEQ ID NO: 89, and CDR-H3 as set forth in SEQ ID NO: 90; (g) CDR-H1 as set forth in SEQ ID NO: 99, CDR-H2 as set forth in SEQ ID NO: 100, and CDR-H3 as set forth in SEQ ID NO: 101; (h) CDR-H1 as set forth in SEQ ID NO: 110, CDR-H2 as set forth in SEQ ID NO: 111, and CDR-H3 as set forth in SEQ ID NO: 112; (i) CDR-H1 as set forth in SEQ ID NO: 121, CDR-H2 as set forth in SEQ ID NO: 122, and CDR-H3 as set forth in SEQ ID NO: 123; (j) CDR-H1 as set forth in SEQ ID NO: 132, CDR-H2 as set forth in SEQ ID NO: 133, and CDR-H3 as set forth in SEQ ID NO: 134; (k) CDR-H1 as set forth in SEQ ID NO: 143, CDR-H2 as set forth in SEQ ID NO: 144, and CDR-H3 as set forth in SEQ ID NO: 145; (l) CDR-H1 as set forth in SEQ ID NO: 154, CDR-H2 as set forth in SEQ ID NO: 155, and CDR-H3 as set forth in SEQ ID NO: 156; (m) CDR-H1 as set forth in SEQ ID NO: 165, CDR-H2 as set forth in SEQ ID NO: 166, and CDR-H3 as set forth in SEQ ID NO: 167; (n) CDR-H1 as set forth in SEQ ID NO: 176, CDR-H2 as set forth in SEQ ID NO: 177, and CDR-H3 as set forth in SEQ ID NO: 178; (o) CDR-H1 as set forth in SEQ ID NO: 187, CDR-H2 as set forth in SEQ ID NO: 188, and CDR-H3 as set forth in SEQ ID NO: 189; (p) CDR-H1 as set forth in SEQ ID NO: 198, CDR-H2 as set forth in SEQ ID NO: 199, and CDR-H3 as set forth in SEQ ID NO: 200; (q) CDR-H1 as set forth in SEQ ID NO: 209, CDR-H2 as set forth in SEQ ID NO: 210, and CDR-H3 as set forth in SEQ ID NO: 211; (r) CDR-H1 as set forth in SEQ ID NO: 220, CDR-H2 as set forth in SEQ ID NO: 221, and CDR-H3 as set forth in SEQ ID NO: 222; (s) CDR-H1 as set forth in SEQ ID NO: 231, CDR-H2 as set forth in SEQ ID NO: 232, and CDR-H3 as set forth in SEQ ID NO: 233; (t) CDR-H1 as set forth in SEQ ID NO: 242, CDR-H2 as set forth in SEQ ID NO: 243, and CDR-H3 as set forth in SEQ ID NO: 244; (u) CDR-H1 as set forth in SEQ ID NO: 253, CDR-H2 as set forth in SEQ ID NO: 254, and CDR-H3 as set forth in SEQ ID NO: 255; (v) CDR-H1 as set forth in SEQ ID NO:264, CDR-H2 as set forth in SEQ ID NO:265, and CDR-H3 as set forth in SEQ ID NO:266; (w) CDR-H1 as set forth in SEQ ID NO: 275, CDR-H2 as set forth in SEQ ID NO: 276, and CDR-H3 as set forth in SEQ ID NO: 276; (x) CDR-H1 as set forth in SEQ ID NO: 286, CDR-H2 as set forth in SEQ ID NO: 287, and CDR-H3 as set forth in SEQ ID NO: 288; (y) CDR-H1 as set forth in SEQ ID NO: 297, and CDR-H3 as set forth in SEQ ID NO:299; (z) CDR-H1 as set forth in SEQ ID NO: 308, CDR-H2 as set forth in SEQ ID NO: 309, and CDR-H3 as set forth in SEQ ID NO: 310; (aa) CDR-H1 as set forth in SEQ ID NO: 319, CDR-H2 as set forth in SEQ ID NO: 320, and CDR-H3 as set forth in SEQ ID NO: 321; (ab) CDR-H1 as set forth in SEQ ID NO: 330, CDR-H2 as set forth in SEQ ID NO: 331, and CDR-H3 as set forth in SEQ ID NO: 332; (ac) CDR-H1 as set forth in SEQ ID NO: 341, CDR-H2 as set forth in SEQ ID NO: 342, and CDR-H3 as set forth in SEQ ID NO: 343; (ad) CDR-H1 as set forth in SEQ ID NO: 352, CDR-H2 as set forth in SEQ ID NO: 353, and CDR-H3 as set forth in SEQ ID NO: 354; (ae) CDR-H1 as set forth in SEQ ID NO: 363, CDR-H2 as set forth in SEQ ID NO: 364, and CDR-H3 as set forth in SEQ ID NO: 365; (af) CDR-H1 as set forth in SEQ ID NO: 374, CDR-H2 as set forth in SEQ ID NO: 375, and CDR-H3 as set forth in SEQ ID NO: 376; (ag) CDR-H1 as set forth in SEQ ID NO: 385, CDR-H2 as set forth in SEQ ID NO: 386, and CDR-H3 as set forth in SEQ ID NO: 386; (ah) CDR-H1 as set forth in SEQ ID NO: 396, CDR-H2 as set forth in SEQ ID NO: 397, and CDR-H3 as set forth in SEQ ID NO: 398; (ai) CDR-H1 as set forth in SEQ ID NO: 407, CDR-H2 as set forth in SEQ ID NO: 408, and CDR-H3 as set forth in SEQ ID NO: 409; (aj) CDR-H1 as set forth in SEQ ID NO: 418, CDR-H2 as set forth in SEQ ID NO: 419, and CDR-H3 as set forth in SEQ ID NO: 420; (ak) CDR-H1 as set forth in SEQ ID NO: 429, CDR-H2 as set forth in SEQ ID NO: 430, and CDR-H3 as set forth in SEQ ID NO: 431; (a1) CDR-H1 as set forth in SEQ ID NO: 440, CDR-H2 as set forth in SEQ ID NO: 441, and CDR-H3 as set forth in SEQ ID NO: 442; (am) CDR-H1 as set forth in SEQ ID NO: 451, CDR-H2 as set forth in SEQ ID NO: 452, and CDR-H3 as set forth in SEQ ID NO: 453; (an) CDR-H1 as set forth in SEQ ID NO: 462, CDR-H2 as set forth in SEQ ID NO: 463, and CDR-H3 as set forth in SEQ ID NO: 464; (ao) CDR-H1 as set forth in SEQ ID NO: 473, CDR-H2 as set forth in SEQ ID NO: 474, and CDR-H3 as set forth in SEQ ID NO: 475; (ap) CDR-H1 as set forth in SEQ ID NO: 484, CDR-H2 as set forth in SEQ ID NO: 485, and CDR-H3 as set forth in SEQ ID NO: 486; (aq) CDR-H1 as set forth in SEQ ID NO: 495, CDR-H2 as set forth in SEQ ID NO: 496, and CDR-H3 as set forth in SEQ ID NO: 497; (ar) CDR-H1 as set forth in SEQ ID NO: 506, CDR-H2 as set forth in SEQ ID NO: 507, and CDR-H3 as set forth in SEQ ID NO: 508; and (as) CDR-H1 as set forth in SEQ ID NO: 517, CDR-H2 as set forth in SEQ ID NO: 518, and CDR-H3 as set forth in SEQ ID NO: 519. a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from: (c) CDR-H1 as set forth in SEQ ID NO:55, CDR-H2 as set forth in SEQ ID NO:56, and CDR-H3 as set forth in SEQ ID NO:57; (n) CDR-H1 as set forth in SEQ ID NO: 176, CDR-H2 as set forth in SEQ ID NO: 177, and CDR-H3 as set forth in SEQ ID NO: 178; (ac) CDR-H1 as set forth in SEQ ID NO: 341, CDR-H2 as set forth in SEQ ID NO: 342, and CDR-H3 as set forth in SEQ ID NO: 343; and (aj) CDR-H1 as set forth in SEQ ID NO: 418, CDR-H2 as set forth in SEQ ID NO: 419, and CDR-H3 as set forth in SEQ ID NO: 420 is preferred, it is also envisaged to provide an antibody construct.

[0031] In said aspect, in the context of the present invention, the first binding domain comprises (a) CDR-L1 as set forth in SEQ ID NO: 36, CDR-L2 as set forth in SEQ ID NO: 37, and CDR-L3 as set forth in SEQ ID NO: 38; (b) CDR-L1 as set forth in SEQ ID NO: 47, CDR-L2 as set forth in SEQ ID NO: 48, and CDR-L3 as set forth in SEQ ID NO: 49; (c) CDR-L1 as set forth in SEQ ID NO:58, CDR-L2 as set forth in SEQ ID NO:59, and CDR-L3 as set forth in SEQ ID NO:60; (d) CDR-L1 as set forth in SEQ ID NO:69, CDR-L2 as set forth in SEQ ID NO:70, and CDR-L3 as set forth in SEQ ID NO:71; (e) CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, and CDR-L3 as set forth in SEQ ID NO: 82; (f) CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93; (g) CDR-L1 as set forth in SEQ ID NO: 102, CDR-L2 as set forth in SEQ ID NO: 103, and CDR-L3 as set forth in SEQ ID NO: 104; (h) CDR-L1 as set forth in SEQ ID NO: 113, CDR-L2 as set forth in SEQ ID NO: 114, and CDR-L3 as set forth in SEQ ID NO: 115; (i) CDR-L1 as set forth in SEQ ID NO: 124, CDR-L2 as set forth in SEQ ID NO: 125, and CDR-L3 as set forth in SEQ ID NO: 126; (j) CDR-L1 as set forth in SEQ ID NO: 135, CDR-L2 as set forth in SEQ ID NO: 136, and CDR-L3 as set forth in SEQ ID NO: 137; (k) CDR-L1 as set forth in SEQ ID NO: 146, CDR-L2 as set forth in SEQ ID NO: 147, and CDR-L3 as set forth in SEQ ID NO: 148; (l) CDR-L1 as set forth in SEQ ID NO: 157, CDR-L2 as set forth in SEQ ID NO: 158, and CDR-L3 as set forth in SEQ ID NO: 159; (m) CDR-L1 as set forth in SEQ ID NO: 168, CDR-L2 as set forth in SEQ ID NO: 169, and CDR-L3 as set forth in SEQ ID NO: 170; (n) CDR-L1 as set forth in SEQ ID NO: 179, CDR-L2 as set forth in SEQ ID NO: 180, and CDR-L3 as set forth in SEQ ID NO: 181; (o) CDR-L1 as set forth in SEQ ID NO: 190, CDR-L2 as set forth in SEQ ID NO: 191, and CDR-L3 as set forth in SEQ ID NO: 192; (p) CDR-L1 as set forth in SEQ ID NO: 201, CDR-L2 as set forth in SEQ ID NO: 202, and CDR-L3 as set forth in SEQ ID NO: 203; (q) CDR-L1 as set forth in SEQ ID NO: 212, CDR-L2 as set forth in SEQ ID NO: 213, and CDR-L3 as set forth in SEQ ID NO: 214; (r) CDR-L1 as set forth in SEQ ID NO: 223, CDR-L2 as set forth in SEQ ID NO: 224, and CDR-L3 as set forth in SEQ ID NO: 225; (s) CDR-L1 as set forth in SEQ ID NO: 234, CDR-L2 as set forth in SEQ ID NO: 235, and CDR-L3 as set forth in SEQ ID NO: 236; (t) CDR-L1 as set forth in SEQ ID NO: 245, CDR-L2 as set forth in SEQ ID NO: 246, and CDR-L3 as set forth in SEQ ID NO: 247; (u) CDR-L1 as set forth in SEQ ID NO: 256, CDR-L2 as set forth in SEQ ID NO: 257, and CDR-L3 as set forth in SEQ ID NO: 258; (v) CDR-L1 as set forth in SEQ ID NO:267, CDR-L2 as set forth in SEQ ID NO:268, and CDR-L3 as set forth in SEQ ID NO:269; (w) CDR-L1 as set forth in SEQ ID NO: 278, CDR-L2 as set forth in SEQ ID NO: 279, and CDR-L3 as set forth in SEQ ID NO: 280; (x) CDR-L1 as set forth in SEQ ID NO: 289, and CDR-L3 as set forth in SEQ ID NO: 291; (y) CDR-L1 as set forth in SEQ ID NO: 300, CDR-L2 as set forth in SEQ ID NO: 301, and CDR-L3 as set forth in SEQ ID NO: 302; (z) CDR-L1 as set forth in SEQ ID NO: 311, CDR-L2 as set forth in SEQ ID NO: 312, and CDR-L3 as set forth in SEQ ID NO: 313; (aa) CDR-L1 as set forth in SEQ ID NO: 322, CDR-L2 as set forth in SEQ ID NO: 323, and CDR-L3 as set forth in SEQ ID NO: 324; (ab) CDR-L1 as set forth in SEQ ID NO: 333, CDR-L2 as set forth in SEQ ID NO: 334, and CDR-L3 as set forth in SEQ ID NO: 335; (ac) CDR-L1 as set forth in SEQ ID NO: 344, CDR-L2 as set forth in SEQ ID NO: 345, and CDR-L3 as set forth in SEQ ID NO: 346; (ad) CDR-L1 as set forth in SEQ ID NO: 355, CDR-L2 as set forth in SEQ ID NO: 356, and CDR-L3 as set forth in SEQ ID NO: 357; (ae) CDR-L1 as set forth in SEQ ID NO: 366, CDR-L2 as set forth in SEQ ID NO: 367, and CDR-L3 as set forth in SEQ ID NO: 368; (af) CDR-L1 as set forth in SEQ ID NO: 377, CDR-L2 as set forth in SEQ ID NO: 378, and CDR-L3 as set forth in SEQ ID NO: 379; (ag) CDR-L1 as set forth in SEQ ID NO: 388, CDR-L2 as set forth in SEQ ID NO: 389, and CDR-L3 as set forth in SEQ ID NO: 390; (ah) CDR-L1 as set forth in SEQ ID NO: 399, CDR-L2 as set forth in SEQ ID NO: 400, and CDR-L3 as set forth in SEQ ID NO: 401; (ai) CDR-L1 as set forth in SEQ ID NO: 410, CDR-L2 as set forth in SEQ ID NO: 411, and CDR-L3 as set forth in SEQ ID NO: 412; (aj) CDR-L1 as set forth in SEQ ID NO: 421, CDR-L2 as set forth in SEQ ID NO: 422, and CDR-L3 as set forth in SEQ ID NO: 423; (ak) CDR-L1 as set forth in SEQ ID NO: 432, CDR-L2 as set forth in SEQ ID NO: 433, and CDR-L3 as set forth in SEQ ID NO: 434; (a1) CDR-L1 as set forth in SEQ ID NO: 443, CDR-L2 as set forth in SEQ ID NO: 444, and CDR-L3 as set forth in SEQ ID NO: 445; (am) CDR-L1 as set forth in SEQ ID NO: 454, CDR-L2 as set forth in SEQ ID NO: 455, and CDR-L3 as set forth in SEQ ID NO: 456; (an) CDR-L1 as set forth in SEQ ID NO: 465, CDR-L2 as set forth in SEQ ID NO: 466, and CDR-L3 as set forth in SEQ ID NO: 467; (ao) CDR-L1 as set forth in SEQ ID NO: 476, CDR-L2 as set forth in SEQ ID NO: 477, and CDR-L3 as set forth in SEQ ID NO: 478; (ap) CDR-L1 as set forth in SEQ ID NO: 487, CDR-L2 as set forth in SEQ ID NO: 488, and CDR-L3 as set forth in SEQ ID NO: 489; (aq) CDR-L1 as set forth in SEQ ID NO: 498, CDR-L2 as set forth in SEQ ID NO: 499, and CDR-L3 as set forth in SEQ ID NO: 500; (ar) CDR-L1 as set forth in SEQ ID NO: 509, CDR-L2 as set forth in SEQ ID NO: 510, and CDR-L3 as set forth in SEQ ID NO: 511; and (as) CDR-L1 as set forth in SEQ ID NO: 520, CDR-L2 as set forth in SEQ ID NO: 521, and CDR-L3 as set forth in SEQ ID NO: 522. a VL region comprising CDR-H1, CDR-L2 and CDR-L3 selected from: (c) CDR-L1 as set forth in SEQ ID NO:58, CDR-L2 as set forth in SEQ ID NO:59, and CDR-L3 as set forth in SEQ ID NO:60; (n) CDR-L1 as set forth in SEQ ID NO: 179, CDR-L2 as set forth in SEQ ID NO: 180, and CDR-L3 as set forth in SEQ ID NO: 181; (ac) CDR-L1 as set forth in SEQ ID NO: 344, CDR-L2 as set forth in SEQ ID NO: 345, and CDR-L3 as set forth in SEQ ID NO: 346; and (aj) CDR-L1 as set forth in SEQ ID NO: 421, CDR-L2 as set forth in SEQ ID NO: 422, and CDR-L3 as set forth in SEQ ID NO: 423. is preferred, it is also envisaged to provide an antibody construct.

[0032] In said aspect, in the context of the present invention, the first binding domain comprises (a) a VL region as set forth in SEQ ID NO: 40 and a VH region as set forth in SEQ ID NO: 39; (b) a VL region as set forth in SEQ ID NO:51 and a VH region as set forth in SEQ ID NO:50; (c) a VL region as set forth in SEQ ID NO:62 and a VH region as set forth in SEQ ID NO:61; (d) a VL region as set forth in SEQ ID NO: 73 and a VH region as set forth in SEQ ID NO: 72; (e) a VL region as set forth in SEQ ID NO: 84 and a VH region as set forth in SEQ ID NO: 83; (f) a VL region as set forth in SEQ ID NO: 95 and a VH region as set forth in SEQ ID NO: 94; (g) a VL region as set forth in SEQ ID NO: 106 and a VH region as set forth in SEQ ID NO: 105; (h) a VL region as set forth in SEQ ID NO: 117 and a VH region as set forth in SEQ ID NO: 116; (i) a VL region as set forth in SEQ ID NO: 128 and a VH region as set forth in SEQ ID NO: 127; (j) a VL region as set forth in SEQ ID NO: 139 and a VH region as set forth in SEQ ID NO: 138; (k) a VL region as set forth in SEQ ID NO: 150 and a VH region as set forth in SEQ ID NO: 149; (l) a VL region as set forth in SEQ ID NO: 161 and a VH region as set forth in SEQ ID NO: 160; (m) a VL region as set forth in SEQ ID NO: 172 and a VH region as set forth in SEQ ID NO: 171; (n) a VL region as set forth in SEQ ID NO: 183 and a VH region as set forth in SEQ ID NO: 182; (o) a VL region as set forth in SEQ ID NO: 194 and a VH region as set forth in SEQ ID NO: 193; (p) a VL region as set forth in SEQ ID NO: 205 and a VH region as set forth in SEQ ID NO: 204; (q) a VL region as set forth in SEQ ID NO: 216 and a VH region as set forth in SEQ ID NO: 215; (r) a VL region as set forth in SEQ ID NO: 227 and a VH region as set forth in SEQ ID NO: 226; (s) a VL region as set forth in SEQ ID NO: 238 and a VH region as set forth in SEQ ID NO: 237; (t) a VL region as set forth in SEQ ID NO: 249 and a VH region as set forth in SEQ ID NO: 248; (u) a VL region as set forth in SEQ ID NO: 260 and a VH region as set forth in SEQ ID NO: 259; (v) a VL region as set forth in SEQ ID NO: 271 and a VH region as set forth in SEQ ID NO: 270; (w) a VL region as set forth in SEQ ID NO: 282 and a VL region as set forth in SEQ ID NO: 281 The VH region of; (x) a VL region as set forth in SEQ ID NO: 293 and a VH region as set forth in SEQ ID NO: 292; (y) a VL region as set forth in SEQ ID NO: 304 and a VH region as set forth in SEQ ID NO: 303; (z) a VL region as set forth in SEQ ID NO: 315 and a VH region as set forth in SEQ ID NO: 314; (aa) a VL region as set forth in SEQ ID NO: 326 and a VH region as set forth in SEQ ID NO: 325; (ab) a VL region as set forth in SEQ ID NO: 337 and a VH region as set forth in SEQ ID NO: 336; (ac) a VL region as set forth in SEQ ID NO: 348 and a VH region as set forth in SEQ ID NO: 347; (ad) a VL region as set forth in SEQ ID NO: 359 and a VH region as set forth in SEQ ID NO: 358; (ae) a VL region as set forth in SEQ ID NO: 370 and a VH region as set forth in SEQ ID NO: 369; (af) a VL region as set forth in SEQ ID NO: 381 and a VH region as set forth in SEQ ID NO: 380; (ag) a VL region as set forth in SEQ ID NO: 392 and a VH region as set forth in SEQ ID NO: 391; (ah) a VL region as set forth in SEQ ID NO: 403 and a VH region as set forth in SEQ ID NO: 402; (ai) a VL region as set forth in SEQ ID NO: 414 and a VH region as set forth in SEQ ID NO: 413; (aj) a VL region as set forth in SEQ ID NO: 425 and a VH region as set forth in SEQ ID NO: 424; (ak) a VL region as set forth in SEQ ID NO: 436 and a VH region as set forth in SEQ ID NO: 435; (a1) a VL region as set forth in SEQ ID NO: 447 and a VH region as set forth in SEQ ID NO: 446; (am) a VL region as set forth in SEQ ID NO: 458 and a VH region as set forth in SEQ ID NO: 457; (an) a VL region as set forth in SEQ ID NO: 469 and a VH region as set forth in SEQ ID NO: 468; (ao) a VL region as set forth in SEQ ID NO: 480 and a VH region as set forth in SEQ ID NO: 479; (ap) a VL region as set forth in SEQ ID NO: 491 and a VH region as set forth in SEQ ID NO: 490; (aq) a VL region as set forth in SEQ ID NO: 502 and a VH region as set forth in SEQ ID NO: 501; (ar) a VL region as set forth in SEQ ID NO: 513 and a VH region as set forth in SEQ ID NO: 512; and (as) a VL region as set forth in SEQ ID NO: 524 and a VH region as set forth in SEQ ID NO: 523 It is also envisaged to provide an antibody construct comprising a VL region and a VH region selected from the group consisting of:

[0033] In said aspect, it is relevant to the present invention that the antibody construct is selected from the group consisting of SEQ ID NOs: 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 18

[0036] It is further envisaged to provide an antibody construct comprising a sequence selected from the amino acid sequences as set forth in any of the following: 4, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514 and 525.

[0034] In said aspect, it is relevant to the present invention that the antibody construct comprises, in order from amino to carboxyl: (a) a first domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 184, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514, and 525; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) a second domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 of WO 2008 / 119567 or as set forth in SEQ ID NO: 15; and (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11, and 12. It is also envisaged to provide an antibody construct comprising:

[0035] In the above aspect, in relation to the present invention, the antibody construct further comprises, in addition to (a) to (d), the following, in order from amino to carboxyl: (e) a first polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17-24; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (g) a second polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17 to 24; It is also envisaged to provide an antibody construct further comprising:

[0036] In said embodiment, in the context of the present invention, SEQ ID NOs: 42, 43, 53, 54, 64, 65, 75, 76, 86, 87, 97, 98, 108, 109, 119, 120, 130, 131, 141, 142, 152, 153, 163, 164, 174, 175, 185, 186, 196, 197, 207, 208, 218, 219, 229, 230, 240, 241, 251, 252, 262, 263, 273, 274, 284, 285, 295, 296, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,

[0036] It is also contemplated to provide an antibody construct having an amino acid sequence selected from the group consisting of: 7, 317, 318, 328, 329, 339, 340, 350, 351, 361, 362, 372, 373, 383, 384, 394, 395, 405, 406, 416, 417, 427, 428, 438, 439, 449, 450, 460, 461, 471, 472, 482, 483, 493, 494, 504, 505, 515, 516, 526 and 527.

[0037] In a second aspect, it is further envisaged in the context of the present invention to provide a polynucleotide encoding the antibody construct of the present invention. In a third aspect, it is also envisaged in relation to the present invention to provide a vector comprising a polynucleotide of the invention.

[0038] In a fourth aspect, it is further envisaged in connection with the present invention to provide a host cell transformed or transfected with the polynucleotide or vector of the invention. In a fifth aspect, it is also envisaged in connection with the present invention to provide a method for the production of an antibody construct of the present invention, the method comprising culturing a host cell of the present invention under conditions allowing expression of the antibody construct and recovering the produced antibody construct from the culture.

[0039] In a sixth aspect, it is further envisaged in the context of the present invention to provide a pharmaceutical composition comprising an antibody construct of the invention or an antibody construct produced according to the method of the invention.

[0040] In said embodiment, it is also envisaged in the context of the present invention that the pharmaceutical composition is stable at about -20°C for at least 4 weeks. It is further envisaged in connection with the present invention to provide an antibody construct of the present invention or an antibody construct produced according to the method of the present invention for use in the prevention, treatment or amelioration of a disease selected from a proliferative disease, a neoplastic disease, a cancer or an immune disorder.

[0041] In said aspect, it is also envisaged in the context of the present invention that the disease is gastrointestinal cancer (eg gastric cancer, esophageal cancer, gastroesophageal cancer or colorectal cancer) or pancreatic cancer. In said aspect, it is also envisaged in the context of the present invention that the disease is gastric cancer.

[0042] In a seventh aspect, it is further envisaged in the context of the present invention to provide a method for the treatment or amelioration of a proliferative disease, a neoplastic disease, cancer or an immune disorder, comprising the step of administering to a subject in need thereof an antibody construct of the present invention or an antibody construct produced according to the method of the present invention, wherein the disease is preferably gastrointestinal or pancreatic cancer, most preferably gastric cancer.

[0043] In an eighth aspect, it is also envisaged in relation to the present invention to provide a kit comprising a construct of the invention or an antibody construct produced according to the method of the invention, a polynucleotide of the invention, a vector of the invention and / or a host cell of the invention.

[0044] In a ninth aspect, it is further envisaged in the context of the present invention to provide a method for the treatment or amelioration of gastrointestinal cancer, the method comprising administering to a subject in need thereof a bispecific antibody construct directed against MUC17 and CD3.

[0045] In a tenth aspect, it is further envisaged in the context of the present invention to provide a bispecific antibody construct directed against MUC17 and CD3 for use in the treatment or amelioration of gastrointestinal cancer. [Brief description of the drawings]

[0046] [Figure 1] Epitope clustering of MUC17 is shown. Epitopes El, E2, E3, E4, E5A and 5B as well as truncated forms of E2 (TR2, TR3, TR4 and TR5 respectively) are marked. Experiments on constructs in which human MUC17 (brown / grey) was replaced by non-functional mouse MUC3 revealed the respective epitopes. 45 MUC-17-scFc bispecific antibody constructs were identified that span the epitope space E2, including the SEA domain. [Diagram 2] MUC17 epitope mapping by on-cell binding of MUC17-scFc bispecific antibody constructs to cells expressing human / mouse chimeric constructs. On-cell binding was assessed by fluorescence activated cell sorting (FACS) and reduced binding to the chimeric constructs indicates that the respective (mutated) domain is essential for MUC-scFc bispecific antibody construct binding. For example, E2 shows reduced binding to the mutants. Thus, E2 is essential for binding to all four tested bispecific antibody constructs. [Diagram 3]MUC17 is expressed in gastric, pancreatic and colorectal cancer cell lines. MUC17 cell surface protein expression was determined by live cell flow cytometry and shown as FACS readout (A). MUC17 mRNA levels in cancer cell lines were determined by quantitative polymerase chain reaction (qPCR). Values ​​are normalized to those of constitutively expressed genes (B). [Figure 4] Cytotoxicity assay against three different MUC17-bearing cell lines with different MUC expression (A: GSU, B: NUGC-4 and C: Ls174T). Constructs tested are: 1=32-G6; 2=1-B6; 3=2-C2 and 4=8-B7. Construct 8-B7 has a slight advantage in terms of cytotoxicity. [Diagram 5] Soluble MUC17 protein (sMUC17, aa 4131-4243 Uniprot) was added to the TDCC assay at 0-1000 ng / ml and the activity of the MUC17-scFc bispecific antibody construct was assessed after 48 h of incubation (target cells GSU (A) or NUGC-4 (B), 10:1 human T cells vs. target cells, Steady Glo readout). Addition of sMUC17 did not affect the cytotoxic activity of the bispecific antibody construct. [Figure 6] MUC17-scFc antibody construct 8-B7 inhibits tumor growth in a xenograft model of colorectal cancer. Female NOD / SCID mice were implanted with 2x106 Ls174T colorectal cancer cells. On day 15, activated T cells expanded to 2x107 were administered by intraperitoneal (IP) injection. MUC17-scFc antibody construct was administered IP on days 16 and 22. Tumor size was measured with a caliper. [Figure 7]A survey of preferred bispecific antibody constructs according to the present invention by group code (optimized library), molecular name, epitope cluster bound by each construct, affinity according to SPR (KD) in [nM], cytotoxic activity (EC50) in NUGC-4 cells in [pM], the ratio (EC50 / KD)*1000 and VH VL configuration. [Figure 8] MUC17-scFc antibody construct 8-B7 has an extended half-life in cynomolgus monkeys (A). Exposure levels are consistent with expected exposure. (B) Cynomolgus monkeys (n=3 per group) were administered 100 mg / kg or 1000 mg / kg MUC17 HLE BiTE® at 0 and 168 hours. Serum was collected at the indicated time points and analyzed for the presence of MUC17 scFc bispecific antibody construct using ELISAs based on either anti-CD3 or anti-MUC17 antibodies. Data were fitted to a two-compartment model. Graphs show individual data (points) and mean values ​​(lines). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] In the context of the present invention, a bispecific antibody construct is provided that specifically targets MUC17 associated with malignant tumors. To this end, first MUC17 is identified as a gene that is upregulated in gastric tumors relative to normal tissue expression. In this regard, MUC17 protein is shown to be expressed in 40-77% of gastric tumors according to immunohistochemistry methods common in the art. MUC17 protein is also demonstrated by flow cytometry to be expressed on the cell surface of gastric and esophageal cancer cell lines, in addition to some pancreatic and colorectal cancer cell lines. Such expression has further been shown to be particularly high in gastric tumors in Chinese patients. Thus, MUC17 is identified as an effective target associated with gastrointestinal cancer, i.e., cancer of the stomach, small intestine and large intestine (colon), esophageal cancer, and pancreatic cancer.

[0048] In the context of the present invention, it is a surprising discovery that the bispecific antibody constructs according to the invention preferably target cancer cells, such as gastric and gastrointestinal cancer cells, which bear MUC17, and in contrast target less non-cancerous cells. MUC17 is normally expressed on the apical surface of non-cancerous intestinal epithelial cells (i.e. located opposite the base of the respective cells) and forms part of the mucosal layer. However, MUC17 is overexpressed in gastric and gastrointestinal cancers, and in such situations is not only restricted to mucosal surfaces but is also expressed on non-mucosal surfaces. Without being bound by theory, it is believed that MUC17 on mucosal surfaces is inaccessible to the bispecific antibody constructs according to the invention, whereas MUC17 expressed on non-mucosal surfaces in cancer cells is more accessible. Thus, the bispecific antibody constructs according to the present invention preferably target MUC17-associated cancer cells and target less non-cancerous cells. This was surprisingly found when comparing the good tolerability in healthy animals with the high anti-tumor efficacy in in vivo cancer models. In particular, immunohistochemistry confirmed MUC17 expression on the mucosal surface of gastrointestinal tissues such as the small intestine taken from monkeys evaluated in exploratory toxicology studies, but advantageously there were no histopathological changes in MUC17-expressing tissues. Good tolerance of non-cancerous cells for the bispecific antibody constructs according to the invention is likewise confirmed in vitro. In contrast, intravenous treatment of tumor-bearing mice with the bispecific antibody constructs according to the invention results in a statistically significant and dose-dependent inhibition of tumor growth compared to placebo-treated mice in the control group. Thus, the bispecific antibody constructs according to the invention are preferably tolerated by patients and preferably feature a well-manageable therapeutic window not previously described for any MUC17-directed agent.

[0049] Bispecific antibody constructs against the EGF-SEA-EGF region of the MUC17 protein are provided in the context of the present invention. Advantageously, targeting this region of the protein results in selectivity from the closest family members (MUC3A, MUC3B, MUC12; e.g., Hollingsworth and Swanson, Nat. Rev. Cancer 2004) and the ability to bind to cell membrane-associated MUC17. MUC17, like other transmembrane mucins, contains a potential cleavage site within the SEA domain.

[0050] Thus, bispecific antibody constructs having a MUC17 EGF-SEA-EGF region and a single chain Fc format that targets CD3 and extends half-life targeting are contemplated herewith. Advantageously, the bispecific antibody constructs of the invention preferably have high affinity (single digit nM K) for target cells bearing the MUC17 target. D ) and potency (<50 pM EC50), allowing for targeting of low or heterogeneous levels of MUC17 in tumor cells of interest.

[0051] Bispecific antibody constructs according to the invention are, for example, cross-reactive to cynomolgus MUC17 (in addition to human MUC17), allowing for non-clinical toxicology testing. The importance of the details of the EGF-SEA-EGF domain sequence of cynomolgus MUC17 is shown for the first time herein.

[0052] In the context of the present invention, it is envisaged that the bispecific antibody construct exhibits binding affinity, potent cytotoxic activity and is the most stable map to the SEA domain. In the context of the present invention, it is envisaged that the bispecific antibody construct has a cysteine ​​clamp, i.e. an intramolecular disulfide bond, in the target binding site for improved stability.

[0053] In the context of the present invention, bispecific antibody constructs provided with a single-chain Fc (scFc) as a half-life extension (HLE) moiety and directed against MUC17 are useful in the treatment of gastric cancer, gastrointestinal cancer, and other gastrointestinal disorders. It is contemplated that the compounds are intended for use in the treatment of gastrointestinal cancer, including colon, esophageal, pancreatic and colorectal cancer.

[0054] Furthermore, in the context of the present invention, it is envisaged that optionally but advantageously, the scFc, i.e. HLE, antibody construct allows for intravenous administration administered only once per week, once every two weeks, once every three weeks or even once every four weeks or less frequently.

[0055] In the context of the present invention, preferred epitopes to be therapeutically targeted are identified by first removing the tandem repeats of MUC17, as they are highly glycosylated and repetitive in sequence. This results, for example, in the underlined region at 376aa and the EGF-like / SEA domain region at 177aa. Advantageously, targeting the EGF-like / SEA domain allows selectivity from closest family members such as MUC3, cross-reactivity with cynomolgus MUC17, and binding to cell membrane-bound MUC17. Subsequently, the inventors generated reagents and assays to assess binding and T cell redirection lysis, activation, and cytokine release. Using these assays, preferred bispecific antibody constructs were confirmed to meet predefined candidate product profiles in terms of affinity, cytotoxic activity, and construct stability.

[0056] To determine the epitope of the preferred bispecific antibody construct directed against MUC17, epitope mapping was performed as described herein. The preferred bispecific antibody construct is directed against the epitope E2, which comprises the SEA domain. The E2 epitope comprises the amino acid (aa) sequence characterized herein as SEQ ID NO: 528. This is essentially the aa sequence of MUC17 according to the uniprot Q685J3 numbering. 4171-4296. Generally, in the context of the present invention, the aa numbering of MUC17 is always made or is intended to be made with reference to the uniprot Q685J3 numbering of MUC17. On the other hand, bispecific antibody constructs targeting the E1 epitope of MUC17, i.e. the epitope N-terminal of the SEA domain (see FIG. 1), surprisingly show undesirable cross-reactivity with MUC3A and MUC3B, which leads to off-target activity and ultimately an increased risk of side effects. Moreover, bispecific antibody constructs directed against epitopes E3 and E4 located at the C-terminus of the SEA domain (see FIG. 1) unexpectedly do not cross-react with cynomolgus MUC17. It is therefore envisaged that the bispecific antibody constructs according to the present invention specifically and exclusively bind to the E2 epitope of MUC17.

[0057] Such preferred bispecific antibody constructs according to the present invention may be further specified based on their structure or their unique detailed epitope binding properties. Preferred bispecific antibody constructs according to the present invention may be determined by calculating a novel indicative ratio of cytotoxicity and affinity as provided herein. For example, said ratio (EC 50 / K D )*1000 is preferably <250. Such a ratio usually indicates good binding to the truncated forms of epitope E2, i.e. TR2 (trunk2: SEQ ID NO: 532) and TR3 (trunk3: SEQ ID NO: 533), whereas a ratio >250 usually indicates good binding to TR2 but not to TR3. In particular, the most preferred constructs usually bind to part of epitope cluster E2 / E5A / 5B and / or TR2 / TR3. They have an (EC 50 :K D)*1000 ratio and belong to related sequence families (e.g., optimized (OPT) library designations 4a, 4b, 5a and 10, whose VH / VL configurations are preferably characterized herein as 4lambda3 or "4I3"). Such constructs are, in the context of the present invention, for example, 8-A7, 8-B7, 8-B8, 8-C7 , 8-H8, 8-D7, 4-E7, 8-F9, 1-A6, 8-H9, 1-B6, 8-F11 and 5-H1. 50 :K D Also preferred are constructs which exhibit a ratio and belong to sequence families (OPT library designations) 1a, 1c and 9. Their VH / VL configuration is characterized as 3 lambda 3 or "3I3". Such constructs are identified in the context of the present invention as, for example, 2-D11, 8-E3, 32-G6, 2-C2, 9-C2, 1-B10, 4-B1, 4-F6, 4-G4, 4-A8, 4-B10, 4-H11 and 4-H2. They bind to epitope clusters E2 / part of E5A / part of E5B and / or part of TR2 / TR3 and have an (EC) of less than about 1500, typically between 250 and 1450. 50 / K D )*1000 ratio and belonging to sequence families (OPT library designations) 6, 7 and 8 are preferred, but less preferred than the two aforementioned sequence families. Their VH / VL configuration is characterized as 2kappa3 or "3k3". Particularly preferred herein are the constructs 32-G6 (SEQ ID NO: 65); 1-B6 (SEQ ID NO: 483); 2-C2 (SEQ ID NO: 428) and 8-B7 (SEQ ID NO: 186). In the context of the present invention, affinity is generally measured by SPC, such as BiacoreB analysis, with results generally given in nM. Cytotoxic activity is generally determined using NUGC-4 cells as MUC17 target cells and unstimulated human PBMCs as CD3 effector cells.

[0058] In the context of the present invention, it is envisaged that preferred bispecific antibody constructs not only exhibit a favourable ratio of cytotoxicity and affinity, but also exhibit sufficient stability properties to facilitate practical handling when formulating, storing and administering said constructs. Sufficient stability is characterised by a high monomer content (i.e. non-aggregated and / or non-associated native molecules) after standard preparation, e.g. at least 65%, more preferably at least 70% and even more preferably at least 75%, as determined by preparative size exclusion chromatography (SEC). Also, the turbidity measured as light absorption at 340 nm, e.g. at a concentration of 2.5 mg / ml, should preferably be 0.025 or less, more preferably 0.020 or less, to conclude an essential lack of undesired aggregates. Advantageously, the high monomer content is maintained after incubation in stress conditions such as freeze / thaw or incubation at 37 or 40° C.

[0059] Thus, the present invention provides The first domain that binds to MUC17, a second domain that binds to an extracellular epitope of the human and macaque CD3 epsilon chain; and optionally a third domain comprising two polypeptide monomers, each of which comprises a hinge, a CH2 domain, and a CH3 domain, the two polypeptide monomers being fused to each other via a peptide linker; The present invention provides an antibody construct comprising:

[0060] In one embodiment, the invention provides a bispecific antibody construct comprising all three such domains. The term "antibody construct" refers to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g. a full-length or complete immunoglobulin molecule. Thus, an antibody construct is capable of binding to its specific target or antigen and / or is derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. Furthermore, a domain that binds to a binding partner according to the invention is herein understood as a binding domain of an antibody construct according to the invention. Typically, a binding domain according to the invention comprises the minimum structural requirements of an antibody that allow target binding. This minimum requirement can be, for example, For example, it may be defined by the presence of at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably all six CDRs. An alternative way of defining the minimal structural requirements of an antibody is the definition of the epitope of the antibody within the structure of a specific target, a protein domain (epitope cluster) of the target protein that constitutes each epitope region, or by reference to a specific antibody that competes with the epitope of the defined antibody. Antibodies on which the constructs according to the invention are based include for example monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies.

[0061] The binding domain of the antibody construct according to the invention may, for example, comprise the CDRs of the above-referenced groups. Preferably, the CDRs are comprised within the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), although it is not necessary that it comprises both. An Fd fragment, for example, has two VH regions and often retains some antigen-binding function of the intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants or binding domains include (1) a Fab fragment, which is a monovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of one arm of an antibody, (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs), and (7) single chain Fvs (scFvs), the latter of which are preferred (e.g., from an scFV library). Exemplary embodiments of antibody constructs according to the invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910 and WO 2015 / 048272.

[0062] The definition of "binding domain" or "domain that binds to" also includes fragments of full-length antibodies such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or "rIgG" ("half antibodies"). Antibody constructs according to the invention may also include modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "multibodies" such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies comprising only one variable domain which may be VHH, VH or VL that specifically binds to an antigen or epitope independent of other V regions or domains.

[0063] As used herein, the term "single-chain Fv", "single-chain antibody" or "scFv" refers to a single polypeptide chain antibody fragment that contains the variable regions from both the heavy and light chains but lacks the constant region. Typically, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains that enables the formation of the desired structure that allows for antigen binding. Single-chain antibodies are described in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore ed. The method is discussed in detail by Pluckthun in 1994, Springer-Verlag, New York, pp. 269-315. Various methods for producing single chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Publication WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, single chain antibodies may be bispecific, multispecific, human and / or humanized, and / or synthetic.

[0064] Furthermore, the definition of the term "antibody construct" includes monovalent, bivalent and polyvalent / multivalent constructs, thus bispecific constructs that specifically bind to only two antigenic structures, as well as polyspecific / multispecific constructs that specifically bind to more than two antigenic structures, e.g., three, four or more, through different binding domains. Furthermore, the definition of the term "antibody construct" includes molecules consisting of only one polypeptide chain as well as molecules consisting of two or more polypeptide chains, where the chains can be either identical (homodimers, homotrimers or homooligomers) or different (heterodimers, heterotrimers or heterooligomers). Examples of the above identified antibodies and variants or derivatives thereof can be found, inter alia, in Harlow and Lane, Antibodies, 19 ... a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Duebel, Antibody Engineering, Springer, 2nd ed. 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0065] As used herein, the term "bispecific" refers to an antibody construct that is "at least bispecific", i.e., it comprises at least a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (herein MUC17MUC17) and the second binding domain binds to another antigen or target (herein CD3). Thus, an antibody construct according to the invention comprises specificity for at least two different antigens or targets. For example, the first domain preferably does not bind to one or more extracellular epitopes of CD3ε of the species described herein. The term "surface antigen of a target cell" refers to an antigenic structure that is expressed by a cell and is present on its cell surface so that it can be accessed by the antibody construct described herein. It may be a protein, preferably an extracellular portion of a protein, or a carbohydrate structure, preferably a carbohydrate structure of a protein such as a glycoprotein. It is preferably a tumor antigen. The term "bispecific antibody construct" according to the present invention also encompasses multispecific antibody constructs, such as trispecific antibody constructs comprising three binding domains or constructs with four or more (e.g. four, five...) specificities.

[0066] When the antibody constructs according to the invention are (at least) bispecific, they do not occur in nature and they differ significantly from naturally occurring products. Thus, a "bispecific" antibody construct or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sites with different specificities. Bispecific antibody constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. For example, see Songsivilai & Lac See Hmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0067] At least two binding domains and a variable domain (VH / VL) of the antibody construct of the present invention may or may not comprise a peptide linker (spacer peptide). The term "peptide linker" according to the present invention comprises an amino acid sequence that interconnects the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antibody construct of the present invention. A peptide linker may also be used to fuse a third domain to another domain of the antibody construct of the present invention. An essential technical feature of such a peptide linker is that it does not contain any polymerization activity. Suitable peptide linkers include those described in US Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344. A peptide linker may also be used to attach other domains or modules or regions (such as half-life extending domains) to the antibody construct of the present invention.

[0068] The antibody construct of the present invention is preferably an "in vitro generated antibody construct". This term refers to an antibody construct according to the above definition, in which all or part of the variable region (e.g. at least one CDR) is generated in non-immune cell selection, such as in vitro phage display, protein chip or any other method that allows testing candidate sequences for antigen binding capacity. Thus, this term preferably excludes sequences that are generated only by genome rearrangement in immune cells of an animal. A "recombinant antibody" is an antibody generated by the use of recombinant DNA technology or genetic engineering.

[0069] As used herein, the term "monoclonal antibody" (mAb) or monoclonal antibody construct refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population that are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site or determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are therefore uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0070] For the preparation of monoclonal antibodies, any technique that results in antibodies produced by continuous cell line culture can be used. For example, the monoclonal antibodies used can be produced by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). Additional examples of techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0071] The hybridomas can then be screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis, e.g., Biacore™, to identify one or more hybridomas that produce an antibody that specifically binds to the designated antigen. Any form of the relevant antigen can be used as the immunogen, for example, recombinant antigen, naturally occurring form, any variant or fragment thereof, as well as antigenic peptides thereof. Surface plasmon resonance, as employed in the Biacore system, can be used to increase the efficiency of binding of phage antibodies to epitopes on surface antigens of target cells (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0072] Another exemplary method for generating monoclonal antibodies includes screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0073] In addition to the use of display libraries, the relevant antigen can be used to immunize a non-human animal, such as a rodent (such as a mouse, hamster, rabbit or rat). In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, mouse strains deficient in mouse antibody production can be engineered with large fragments of the human Ig (immunoglobulin) locus. Hybridoma technology can be used to generate and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. See, for example, XENOMOUSE™, Green et al. (1994) Nature 1999, 14:1311-1325. See Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, WO 96 / 34096 and WO 96 / 33735.

[0074] Monoclonal antibodies, once obtained from a non-human animal, can also be modified, e.g., humanized, deimmunized, chimerized, etc., using recombinant DNA techniques known in the art. Examples of modified antibody constructs include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with altered effector functions (see, e.g., U.S. Pat. No. 5,648,260; Kontermann et al., supra). (see, for example, Little (2009) and Duebel (2010).

[0075] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for antigens during an immune response. Repeated exposure to the same antigen leads the host to produce antibodies with successively increasing affinities. As with natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations within the CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling methods. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range.

[0076] A preferred type of amino acid substitution variant of an antibody construct is one that involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such substitution variants includes affinity maturation using phage display. Briefly described and several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants so generated are displayed in a monovalent form from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the binding domain and, for example, human MUC17. Such contact and adjacent residues are candidates for substitution by techniques detailed herein. After generating such variants, a panel of variants may be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0077] The monoclonal antibodies and antibody constructs of the present invention include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences. Various methods for producing chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0078] Antibodies, antibody constructs, antibody fragments or antibody variants may also be modified by specific deletion of human T-cell epitopes (a method called "deimmunization") by the methods disclosed in the Examples of WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of the antibody can be analyzed for peptides that bind to MHC class II. These peptides represent potential T-cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). For the detection of potential T-cell epitopes, a computer modeling approach called "peptide threading" can be applied, as described in WO 98 / 52976 and WO 00 / 34317, and in addition, a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, and therefore represent potential T cell epitopes. Potential T cell epitopes detected can be eliminated by substituting a small number of amino acid residues in the variable domains, or preferably by single amino acid substitutions. Conservative substitutions are usually made. In many, but not all, amino acids common to positions in human germline antibody sequences can be used. For human germline sequences, see, e.g., Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16(5):237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (Tomlinson, LA. et al. MRC Centre (Edited by the American Society for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, e.g., for the framework regions and CDRs. For example, the consensus human framework regions described in U.S. Patent No. 6,300,064 can be used.

[0079] A "humanized" antibody, antibody construct, variant or fragment thereof (Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody) is an antibody or immunoglobulin of largely human sequence that contains minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also referred to as CDR) of the recipient are replaced by residues from a hypervariable region of a non-human (e.g. rodent) species (donor antibody) such as mouse, rat, hamster or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may also comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0080] Humanized antibodies or fragments thereof can be generated by replacing sequences of Fv variable domains not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214, and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, 5,859,205, and 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences that encode all or part of an immunoglobulin Fv variable domain from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas and other sources that produce antibodies against a predetermined target as described above. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0081] Humanized antibodies can also be produced using transgenic animals, such as mice, that express human heavy and light chain genes but are unable to express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting method that can be used to prepare the humanized antibodies described herein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular human antibody can be replaced with at least a portion of a non-human CDR, or only a portion of the CDRs can be replaced with a non-human CDR. It is only necessary to replace as many CDRs as are required for the binding of the humanized antibody to a given antigen.

[0082] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such modified immunoglobulin molecules can be produced by any of several techniques known in the art (see, for example, Teng et al., Proc. Natl. Acad. Sci. USA, 80:73-74). 08-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982 and European Patent No. 239400).

[0083] The terms "human antibody," "human antibody construct," and "human binding domain" include antibodies, antibody constructs, and binding domains having antibody regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) supra. A human antibody, antibody construct, or binding domain of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly CDR3. A human antibody, antibody construct, or binding domain may have at least one, two, three, four, five, or more positions substituted with an amino acid residue not encoded by human germline immunoglobulin sequences. As used herein, the definitions of human antibody, antibody construct and binding domain also contemplate fully human antibodies that contain only human sequences of non-artificially and / or genetically modified antibodies, which can be obtained by using techniques or systems such as Xenomouse. Preferably, a "fully human antibody" does not contain amino acid residues that are not encoded by human germline immunoglobulins.

[0084] In some embodiments, the antibody construct of the present invention is an "isolated" or "substantially pure" antibody construct. When used to describe the antibody constructs disclosed herein, "isolated" or "substantially pure" refers to an antibody construct that has been identified, separated and / or recovered from components of its production environment. Preferably, the antibody construct is free or substantially free of association with all other components from its production environment. Contaminating components of its production environment, such as components arising from recombinant transfected cells, are materials that would normally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. The antibody construct may, for example, comprise at least about 5% or at least about 50% by weight of the total protein in a given sample. It is understood that an isolated protein may comprise 5% to 99.9% by weight of the total protein content, depending on the circumstances. The polypeptide may be produced at significantly higher concentrations by the use of inducible promoters or high expression promoters, such that the polypeptide is produced at high concentration levels. This definition includes production of antibody constructs in a variety of organisms and / or host cells known in the art. In a preferred embodiment, the antibody construct will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie blue or, preferably, silver staining. Typically, however, an isolated antibody construct will be prepared by at least one purification step.

[0085] The term "binding domain" in the context of the present invention characterizes a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target site on a target molecule (antigen), here MUC17 and CD3, respectively. The structure and function of the first binding domain (recognizing MUC17) and preferably also the structure and / or function of the second binding domain (recognizing CD3) are based on the structure and / or function of an antibody, e.g. a full length or complete immunoglobulin molecule, and / or are derived from the variable heavy (VH) and / or variable light (VL) chain domains of an antibody or a fragment thereof. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). The second binding domain preferably confers target binding. It also includes the minimal structural requirements of an antibody that allows for the synthesis of a second binding domain. More preferably, the second binding domain comprises at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). It is envisaged that the first and / or second binding domains are generated or obtained by phage display or library screening methods other than grafting CDR sequences from an existing (monoclonal) antibody onto a scaffold.

[0086] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may contain proteinaceous and non-proteinaceous portions (e.g. chemical linkers or chemical cross-linking agents such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments and peptides, usually having less than 30 amino acids) comprise two or more amino acids linked together through covalent peptide bonds (resulting in a chain of amino acids).

[0087] As used herein, the term "polypeptide" refers to a group of molecules that usually consists of more than 30 amino acids. Polypeptides may further form multimers, such as dimers, trimers and higher oligomers, i.e., consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher order structures of such multimers are therefore referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its native form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide", "polypeptide" and "protein" also refer to naturally modified peptides / polypeptides / proteins, which are modified, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. As referred to herein, "peptide", "polypeptide" or "protein" may also be chemically modified, such as pegylation. Such modifications are well known in the art and are described herein below.

[0088] Preferably, the binding domain that binds MUC17 and / or the binding domain that binds CD3ε are human binding domains. Antibodies and antibody constructs that include at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs with non-human variable and / or constant regions, such as from rodents (e.g., mice, rats, hamsters or rabbits). The presence of such rodent-derived proteins may result in rapid clearance of the antibody or antibody construct or may generate an immune response against the antibody or antibody construct by the patient. To avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be generated by introducing human antibody functions into rodents such that the rodent produces fully human antibodies.

[0089] The ability to clone and reconstruct megabase-sized human loci in yeast artificial chromosomes YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents will provide unique insight into the expression and regulation of nascent human gene products, their transfer to other systems, and their involvement in disease induction and progression.

[0090] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B cell development. Moreover, such a strategy could provide insight into the role of Ig loci in human diseases. This would provide an ideal source for the generation of fully human monoclonal antibodies (mAbs), which would be an important milestone in realizing the potential of antibody therapy. Fully human antibodies or antibody constructs are expected to minimize the immunogenic and allergic reactions inherent to mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of the administered antibodies / antibody constructs. The use of fully human antibodies or antibody constructs can be expected to provide significant advantages in the treatment of chronic and recurrent human diseases, such as inflammation, autoimmunity and cancer, which require repeated administration of compounds.

[0091] One approach towards this goal has been to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci, with the expectation that such mice would produce a broad repertoire of human antibodies without producing mouse antibodies. The large human Ig fragments would preserve the broad diversity of variable genes and the proper regulation of antibody production and expression. By utilizing the mouse machinery for antibody diversification and selection and the lack of immune tolerance to human proteins, the human antibody repertoire recapitulated in this mouse strain should produce high affinity antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with the desired specificity could be easily generated and selected. This general strategy was demonstrated in conjunction with the generation of the first XenoMouse mouse strains (see Green et al. Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered with YACs containing 245 kb and 190 kb sized germline configured fragments of the human heavy and kappa light chain loci, respectively, that contained the core sequences of the variable and constant regions. The human Ig-containing YACs proved compatible with the mouse system for both antibody rearrangement and expression, and were able to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and to produce antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing multiple V genes, additional regulatory elements, and human Ig constant regions, could recapitulate a virtually complete repertoire characterized by the human humoral response to infection and immunization. Recently, extending the work of Green et al., the introduction of megabase sized germline configured YAC fragments of the human heavy and kappa light chain loci, respectively, introduced approximately 80% of the human antibody repertoire. See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application Publication No. 08 / 759,620.

[0092] The generation of XenoMouse animals has been described in U.S. Patent Application Publication Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, 08 / 463,191, 08 / 462,837, 08 / 463,192, 08 / 463,281, 08 / 463,306, 08 / 463,193, 08 / 463,282, 08 / 463,309, 08 / 463,308, 08 / 463,309 ... The present invention is further discussed and detailed in U.S. Patent Nos. 08 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752 and 08 / 759,620; and U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181 and 5,939,598, and Japanese Patent Nos. 3068180B2, 3068506B2 and 3068507B2. Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), European Specific See also Patent Publication No. 0463151B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310 and WO 03 / 47336.

[0093] In another approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region and a second constant region (preferably a gamma constant region) form a construct that is inserted into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; and 5,877,397 to Lonberg and Kay, respectively. Nos. 5,874,299; and 6,255,458, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205; 5,721,367; and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharm and U.S. Patent Application Publication Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International.See also EP 0546073B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, and U.S. Pat. No. 5,981,175. See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0094] Kirin has also demonstrated the production of human antibodies from mice into which large chromosome pieces or entire chromosomes have been introduced by microcell fusion. See EP 773288 and EP 843961. Xenerex Biosciences is developing a promising human antibody production technology in which SCID mice are reconstituted with human lymphoid cells, e.g., B and / or T cells. The mice are then immunized with an antigen and allowed to generate an immune response against that antigen. See U.S. Pat. Nos. 5,476,996; 5,698,767; and 5,958,765.

[0095] The human anti-mouse antibody (HAMA) response has led industry to produce chimeric or otherwise humanized antibodies. However, certain human anti-chimeric antibody (HACA) responses remain. are expected to be observed, particularly with chronic or multiple dose antibody use. It would therefore be desirable to provide an antibody construct that includes a human binding domain for MUC17 and a human binding domain for CD3ε to eliminate the concerns and / or impact of HAMA or HACA reactions.

[0096] The terms "(specifically) bind", "(specifically) recognize", "(specifically) induce" and "(specifically) react" mean, according to the present invention, that a binding domain interacts or specifically interacts with a target molecule (antigen), here a given epitope or a given target site on MUC17 and CD3ε, respectively.

[0097] The term "epitope" refers to a site on an antigen to which a binding domain, such as an antibody or immunoglobulin or a derivative, fragment or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and thus the term epitope is also sometimes referred to herein as an "antigenic structure" or "antigenic determinant". The binding domain is therefore the "antigen interaction site". Said binding / interaction is understood to also define "specific recognition".

[0098] An "epitope" can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. A "linear epitope" is an epitope that comprises an epitope in which a primary amino acid sequence is recognized. A linear epitope usually comprises at least 3 or at least 4, and more usually at least 5, or at least 6, or at least 7, e.g., about 8 to about 10 amino acids in a unique sequence.

[0099] A "conformational epitope", in contrast to a linear epitope, is an epitope in which the primary sequence of amino acids that comprise the epitope is not the only element that defines the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the binding domain). In general, a conformational epitope comprises a larger number of amino acids compared to a linear epitope. With respect to the recognition of a conformational epitope, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is contained within the surface antigen protein of a target cell). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, thereby allowing the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, and site-specific spin labeling and electron paramagnetic resonance (EPR) spectroscopy.

[0100] Methods for epitope mapping are described below. When a region (a contiguous stretch of amino acids) of human MUC17 protein is exchanged or substituted with the corresponding region of non-human and non-primate MUC17 (e.g. mouse MUC17, but also chicken, rat, hamster, rabbit, etc.), a reduction in binding of the binding domain is expected to occur, provided that the binding domain is not cross-reactive with the non-human, non-primate MUC17 used. Said reduction is preferably at least 10%, 20%, 30%, 40% or 50%; more preferably at least 60%, 70% or 80% and most preferably 90%, 95% or even 100% compared to the binding to the corresponding region in the human MUC17 protein, assuming that the binding to the corresponding region of the human MUC17 protein is 100%. It is envisaged that the above human MUC17 / non-human MUC17 chimeras are expressed in CHO cells. It is also envisioned that human MUC17 / non-human MUC17 chimeras are fused to the transmembrane and / or cytoplasmic domains of different membrane-associated proteins, such as EpCAM.

[0101] In an alternative or additional method of epitope mapping, several truncations of the human MUC17 extracellular domain can be made to determine the specific regions recognized by the binding domain. In these truncations, different extracellular MUC17 domains / subdomains or regions are deleted stepwise starting from the N-terminus. It is envisaged that truncated MUC17 can be expressed in CHO cells. It is also envisaged that truncated MUC17 may be fused to the transmembrane and / or cytoplasmic domains of different membrane-associated proteins such as EpCAM. It is also envisaged that truncated MUC17 may include a signal peptide domain at their N-terminus, e.g. a signal peptide derived from mouse IgG heavy chain signal peptide. It is further envisaged that truncated MUC17 may include a v5 domain at the N-terminus (following the signal peptide) that can confirm their correct expression on the cell surface. It is expected that reduced or lost binding occurs for truncated MUC17 that no longer includes the MUC17 region recognized by the binding domain. The reduction in binding is preferably at least 10%, 20%, 30%, 40% or 50%; more preferably at least 60%, 70%, 80% and most preferably 90%, 95% or even 100%, where binding to the entire human MUC17 protein (or its extracellular region or domain) is taken as 100%.

[0102] A further method for determining the contribution of specific residues of MUC17 to recognition by an antibody construct or binding domain is alanine scanning (see, e.g., Morrison KL & Weiss GA. Cur Opin Chem Biol. 2001 Jun;5(3):302-7), in which each residue analyzed is replaced with alanine, for example by site-directed mutagenesis. Alanine is used because it is not bulky, is chemically inert, and yet has a methyl functionality that mimics the secondary structure criteria that many other amino acids have. If it is desirable to preserve the size of the residue to be mutated, sometimes bulky amino acids such as valine or leucine can be used. Alanine scanning is a mature technique that has been used for a long time.

[0103] The interaction between a binding domain and an epitope or an epitope-containing region means that the binding domain exhibits a measurable affinity for the epitope / epitope-containing region on a particular protein or antigen (herein MUC17 and CD3, respectively), and generally does not exhibit significant reactivity with proteins or antigens other than MUC17 or CD3. "Measurable affinity" refers to a binding domain that exhibits a measurable affinity of about 10 -6 M(KD) or stronger. Preferably, the binding affinity is about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9 M, the binding is considered specific. Whether a binding domain specifically reacts with or binds to a target can be easily tested, inter alia, by comparing the reaction of said binding domain to a target protein or antigen with the reaction of said binding domain to proteins or antigens other than MUC17 or CD3. Preferably, the binding domain of the invention essentially or substantially does not bind to proteins or antigens other than MUC17 or CD3 (i.e. the first binding domain cannot bind to proteins other than MUC17 and the second binding domain cannot bind to proteins other than CD3). It is an envisaged feature of the antibody construct according to the invention to have superior affinity properties compared to other HLE formats. Such superior affinity consequently suggests an extended half-life in vivo. The longer half-life of the antibody construct according to the invention may reduce the duration and frequency of administration, which usually contributes to improved patient compliance. This is particularly important, since the antibody construct of the invention is particularly beneficial for cancer patients who are highly debilitated or even multi-disease.

[0104] The terms "essentially / substantially not bound" or "cannot be bound" are used to describe the results of the present invention. This means that the binding domain does not bind to proteins or antigens other than MUC17 or CD3, i.e., when the binding to MUC17 or CD3, respectively, is taken as 100%, it does not show more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6% or 5% reactivity to proteins or antigens other than MUC17 or CD3.

[0105] Specific binding is believed to be brought about by specific motifs in the amino acid sequences of the binding domain and the antigen. Thus, binding occurs as a result of their primary, secondary and / or tertiary structure as well as secondary modifications of said structure. Specific interaction of the antigen interaction site with its specific antigen may result in simple binding of said site to the antigen. Furthermore, specific interaction of the antigen interaction site with its specific antigen may alternatively or additionally result in the initiation of a signal, for example by inducing a conformational change in the antigen, oligomerization of the antigen, etc.

[0106] The term "variable" refers to that portion of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability in sequence and is involved in determining the specificity and binding affinity of a particular antibody. The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single antigen-binding site.

[0107] The variability is not uniformly distributed throughout the variable domain of an antibody, but is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e. non-hypervariable) parts of the variable domains are called "framework" regions (FRMs or FRs) and provide a scaffold for the six CDRs in the three-dimensional space that forms the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3 and FR4) that largely adopt a β-sheet configuration, which are connected by three hypervariable regions that form loop connections and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRMs and contribute to the formation of the antigen-binding site with the hypervariable regions of the other chain (see Kabat et al., supra).

[0108] The term "CDR" and its plural "CDRs" refer to the complementarity determining regions, three of which constitute the binding properties of the light chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three of which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3). The CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen and thus contribute to the functional activity of the antibody molecule. That is, they are the primary determinants of antigen specificity.

[0109] The exact definition of the boundaries and lengths of CDRs are subject to various classifications and numbering systems. Thus, CDRs may be represented by Kabat, Chothia, contact, or any other boundary definition, including the numbering systems described herein. Although the boundaries are different, each of these systems has some overlap in the parts that constitute the so-called "hypervariable regions" in the variable sequences. Thus, the definitions of CDRs according to these systems may differ in length and in the boundaries with respect to the adjacent framework regions. See, for example, Kabat (an approach based on interspecies sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol, 1987, 196:901-917; and MacCallum et al., J. Mol. Biol, 1996, 262:732). Yet another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, e.g., Protein Sequence and Struct See, for example, Nature Analysis of Antibody Variable Domains. In:Antibody Engineering Lab Manual (Ed.:Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). To the extent that two residue identification techniques define overlapping regions rather than identical regions, they can be combined to define hybrid CDRs. However, numbering according to the so-called Kabat system is preferred.

[0110] Usually, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main chain conformation that the antigen-binding (CDR) loop adopts. From comparative structural studies, it has been found that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Thus, corresponding loops between antibodies can have very similar three-dimensional structures, despite the high amino acid sequence variability found in most of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J. Mol. Biol, 1996, 263:800). Furthermore, there is a relationship between the adopted loop structure and its surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and by the amino acid residues present at key positions within the loop as well as within the conserved framework (i.e., outside the loop), and therefore assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0111] The term "canonical structure" may also include consideration of the linear sequence of an antibody, for example, as classified by Kabat (Kabat et al., supra). The Kabat numbering scheme is a widely adopted standard for numbering the amino acid residues of antibody variable domains in a consistent manner, and is the preferred scheme for application in the present invention, as mentioned elsewhere herein. Additional structural considerations may also be used to determine the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering system may be described by the Chothia et al. numbering system and / or may be revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence may be classified into a canonical class that allows, among other things, the identification of a suitable chassis sequence (e.g., based on the desire to include various canonical structures in a library). The Kabat numbering system and the Chothia numbering system of the amino acid sequence of an antibody may be used to determine the canonical structure of an antibody. The structural considerations described in Harlow et al., supra, and their significance for interpreting the canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known in the art. For a general review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0112] The CDR3 of the light chain and especially the CDR3 of the heavy chain may be the most important determinant of antigen binding within the light and heavy chain variable regions.In some antibody constructs, the heavy chain CDR3 is likely to be the main contact area between antigen and antibody.An in vitro selection scheme that changes only CDR3 can be used to change the binding properties of the antibody or to determine which residues contribute to antigen binding.Therefore, CDR3 is usually the greatest source of molecular diversity within the antibody binding site.For example, H3 can be as short as 2 amino acid residues or more than 26 amino acids.

[0113] In a classical full-length antibody or immunoglobulin, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, while the two H chains are linked together in a single covalent disulfide bond depending on the H chain isotype. The VH domains are linked to each other by one or more disulfide bonds. The CH domain closest to VH is usually referred to as CH1. The constant ("C") domains are not directly involved in antigen binding, but exhibit various effector functions such as antibody-dependent, cell-mediated cytotoxicity, and complement activation. The Fc region of an antibody is contained within the heavy chain constant domain and can interact with Fc receptors located on the cell surface, for example.

[0114] The sequences of antibody genes after construction and somatic mutation are highly diverse, and these diversified genes are 10 It is predicted that these genes encode different antibody molecules (Immunoglobulin Genes, 2 nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived in whole or in part from at least one sequence encoding at least one immunoglobulin. The sequence may be generated by in vivo rearrangement of the V, DJ segments of the heavy chain and the V and J segments of the light chain. Alternatively, the sequence may be generated from a cell in response to, for example, an in vitro stimulus that causes the rearrangement. Alternatively, some or all of the sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods (see, for example, U.S. Pat. No. 5,565,332). A repertoire may include only one sequence or may include multiple sequences, including those in a genetically diverse collection.

[0115] The term "Fc portion" or "Fc monomer" in the context of the present invention means a polypeptide comprising at least one domain with the function of a CH2 domain and at least one domain with the function of a CH3 domain of an immunoglobulin molecule. As is evident from the term "Fc monomer", a polypeptide comprising these CH domains is a "polypeptide monomer". An Fc monomer may be a polypeptide comprising a fragment of an immunoglobulin constant region excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but maintaining at least a functional part of one CH2 domain and a functional part of one CH3 domain, the CH2 domain being amino-terminal to the CH3 domain. In a preferred embodiment of this definition, an Fc monomer may be a polypeptide constant region comprising a part of an Ig-Fc hinge region, a CH2 region and a CH3 region, the hinge region being amino-terminal to the CH2 domain. It is envisaged that the hinge region of the present invention promotes dimerization. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by papain digestion of an immunoglobulin region (which of course results in a dimer of two Fc polypeptides). In another aspect of this definition, an Fc monomer can be a polypeptide region that includes a CH2 region and a portion of a CH3 region. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by pepsin digestion of an immunoglobulin molecule. In one embodiment, the polypeptide sequence of an Fc monomer is substantially similar to the Fc polypeptide sequence of an IgG1Fc region, an IgG2Fc region, an IgG3Fc region, an IgG4Fc region, an IgMFc region, an IgAFc region, an IgDFc region, and an IgEFc region (see, for example, Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because there is some variation between immunoglobulins, and simply for clarity, the Fc monomer refers to the last two heavy chain constant region immunoglobulin domains of IgA, IgD and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM. As mentioned, the Fc monomer may also include a flexible hinge N-terminal to these domains. In the case of IgA and IgM, the Fc monomer may include a J chain.For IgG, the Fc portion comprises immunoglobulin domains CH2 and CH3 and the hinge between the first two domains and CH2. Although the boundaries of the Fc portion may vary, an example of a human IgG heavy chain Fc portion comprising functional hinge, CH2 and CH3 domains, for example, begins at the carboxyl terminus of the CH3 domain, from residues D231 (of the hinge domain - corresponding to D234 in Table 1 below) to P476, L476 (for IgG4) according to the numbering according to Kabat. Two Fc moieties or Fc monomers fused to each other via a peptide linker define the third domain of the antibody construct of the invention, which may also be defined as an scFc domain.

[0116] In one embodiment of the invention, it is envisaged that the scFc domains disclosed herein, the Fc monomers each fused to one another, are comprised only in the third domain of the antibody construct.

[0117] In accordance with the present invention, IgG hinge regions may be identified by similarity with the Kabat numbering set out in Table 1. In accordance with the above, it is envisaged that for the hinge domain / region of the present invention, the minimum requirement comprises amino acid residues corresponding to the stretch of IgG1 sequence from D231 D234 to P243 according to the Kabat numbering. It is likewise envisaged that the hinge domain / region of the present invention comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 477) (corresponding to the stretch D234 to P243 shown in Table 1 below - variants of said sequence are also envisaged, as long as the hinge region still promotes dimerization). In a preferred embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain within the third domain of the antibody construct is removed by a N314X substitution, where X is any amino acid that is not Q. Said substitution is preferably a N314G substitution. In a more preferred embodiment, said CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0118] It is also envisaged that the third domain of the antibody construct of the invention comprises or consists of, in amino to carboxyl order: DKTHTCPPCP (SEQ ID NO: 477) (i.e. hinge)-CH2-CH3-linker-DKTHTCPPCP (SEQ ID NO: 477) (i.e. hinge)-CH2-CH3. The peptide linker of said antibody construct is in a preferred embodiment characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 1) or a polymer thereof, i.e. (Gly4Ser)x, where x is an integer of 5 or more (e.g. 5, 6, 7, 8 etc. or more), with 6 being preferred ((Gly4Ser)6). The construct may further comprise the substitutions mentioned above: N314X, preferably N314G and / or the further substitutions V321C and R309C. In a preferred embodiment of the inventive antibody construct as defined herein above, it is envisaged that the second domain binds to an extracellular epitope of the human and / or macaque CD3 epsilon chain.

[0119] Table 1: Kabat numbering of amino acid residues in the hinge region

[0120] [Table 1]

[0121] In a further embodiment of the invention, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 478), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 479) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 486) and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 480). The IgG1 subtype hinge sequence may be the following sequence EPKSCDKTHTCPPCP (as shown in Table 1 and in SEQ ID NO: 487). Thus, these core hinge regions are also envisaged in the context of the present invention.

[0122] The locations and sequences of IgG CH2 and IgG CD3 domains can be identified by similarity using the Kabat numbering set forth in Table 2.

[0123] [Table 2]

[0124] In one embodiment of the invention, the amino acid residues highlighted in bold in the CH3 domain of the first or both Fc monomers are deleted. The peptide linker by which the polypeptide monomers of the third domain ("Fc portion" or "Fc monomers") are fused to one another preferably comprises at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker comprises at least Preferably, the linker comprises at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). It is also preferred that the linker comprises at most 40 amino acid residues, more preferably at most 35 amino acid residues, most preferably exactly 30 amino acid residues. A preferred embodiment of such a peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 1), or a polymer thereof, i.e. (Gly4Ser)x, where x is an integer of 5 or more (e.g. 6, 7 or 8). Preferably, the integer is 6 or 7, more preferably the integer is 6.

[0125] When a linker is used to fuse the first domain with the second domain, or the first or second domain with the third domain, this linker is preferably of sufficient length and sequence to ensure that each of the first and second domains retains their different binding specificities independently of each other. In the case of peptide linkers connecting at least two binding domains (or two variable domains) in the antibody construct of the present invention, these peptide linkers are preferably those that contain only a few amino acid residues, for example those that contain 12 amino acid residues or less. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. Conceivable peptide linkers with less than 5 amino acids contain 4, 3, 2 or 1 amino acid, with Gly-rich linkers being preferred. A preferred embodiment of a peptide linker for the fusion of the first and second domains is shown in SEQ ID NO: 1. A preferred linker embodiment of a peptide linker for the fusion of the second and third domains is the (Gly)4-linker, also called G4-linker.

[0126] A particularly preferred "single" amino acid in connection with one of the above "peptide linkers" is Gly. Thus, the above peptide linker may consist of a single amino acid Gly. In a preferred embodiment of the invention, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 1) or a polymer thereof, i.e. (Gly4Ser)x, where x is an integer of 1 or more (e.g. 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1-12. The characteristics of said peptide linkers, including not promoting secondary structures, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Moreover, peptide linkers that do not promote any secondary structures are preferred. The mutual linkage of the above domains can be provided, for example, by genetic engineering as described in the Examples. Methods for preparing fused and operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (see, for example, WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2003). Laboratory Press,Cold Spring Harbor,New York, 2001).

[0127] In an antibody construct or preferred embodiment of the invention, the first domain and the second domain form an antibody construct of a format selected from the group consisting of: (scFv)2, scFv-single domain mAb, diabody and oligomers of any of these formats.

[0128] According to a particularly preferred embodiment, and as described in the accompanying examples, the first and second domains of the antibody construct of the invention are "bispecific single chain antibody constructs", more preferably bispecific "single chain Fvs" (scFvs). Fv fragments Although the two domains, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker as described above herein, which allows them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as complete or full-length antibodies. Thus, a single-chain variable fragment (scFv) is a fusion protein of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an immunoglobulin, usually connected by a short linker peptide of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can either link the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0129] Bispecific single chain antibody constructs are known in the art and have been described in WO 99 / 54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loeffler, Blood, (2000), 95, 6, 2098-2103; Bruehl, Immunol., (2001), 166, 2420-2426; Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for the production of single chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778; Kontermann and Duebel (2010), supra; and Little (2009), supra) can be adapted to generate single chain antibody constructs that specifically recognize a target of choice.

[0130] Bivalent (also called divalent) or bispecific single chain variable fragments (bi-scFv or di-scFv with the format (scFv)2) can be created by linking two scFv molecules (e.g., using a linker as described herein above). If these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably called bivalent (i.e., it has a valency of two for the same target epitope). If these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably called bispecific. Linking can be done by creating a single peptide chain with two VH and two VL regions to generate tandem scFvs (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules with a linker peptide that is too short (e.g., about 5 amino acids) to allow the two variable regions to fold together, allowing the scFvs to dimerize, a form known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0131] In accordance with the present invention, either the first domain, the second domain or the first and second domains may comprise a single domain antibody, a variable domain of a single domain antibody or at least the CDRs of a single domain antibody, respectively. A single domain antibody is an antibody that contains only one (monomeric) antibody variable domain that is capable of selectively binding to a specific antigen independently of other V regions or domains. The first single domain antibodies were generated from heavy chain antibodies found in camels, which contain V H The H fragment is also called the V fragment. NARSingle domain antibodies, called fragments, can be obtained. An alternative approach is to split the dimeric variable domain from a common immunoglobulin, for example from human or rodent origin, into monomers, thereby obtaining VH or VL as single domain Abs. Most research on single domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind target epitopes. Examples of single domain antibodies are called sdAbs, nanobodies or single variable domain antibodies.

[0132] Therefore, (single domain mAb)2 is V H , V L , V H H and V NAR A monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies individually selected from the group comprising: a) a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies individually selected from the group comprising: a) a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies individually selected from the group comprising: a) a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies individually selected from the group comprising: a) a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies individually selected from the group comprising: a) a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies individually selected from the group comprising:

[0133] Whether an antibody construct binds in competition with another given antibody construct can be measured by a competitive assay, such as a competitive ELISA or a cell-based competitive assay. Avidin-conjugated microparticles (beads) can also be used. Each of these beads can be used as a substrate when reacting with biotinylated proteins, as with an avidin-coated ELISA plate, on which the assay can be performed. The antigen is coated on the beads, and then pre-coated with the primary antibody. A secondary antibody is added to confirm any further binding. Possible means for reading include flow cytometry.

[0134] T cells or T lymphocytes are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. There are several subsets of T cells, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of T cell receptors (TCRs) on their cell surface. The TCR is responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules and is composed of two different protein chains. In 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain. When the TCR binds to an antigen peptide and MHC (peptide / MHC complex), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules and activated or released transcription factors.

[0135] The CD3 receptor complex is a protein complex and is composed of four chains. In mammals, this complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor CD3 complex, which generates activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta) and CD3ε (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor tyrosine-based activation motif, or ITAM for short, which is essential for the signaling ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene present on chromosome 11 in humans. The most preferred CD3 epsilon epitope is included in the range of amino acid residues 1 to 27 of the human CD3 epsilon extracellular domain. The antibody constructs according to the invention typically and advantageously exhibit only low levels of undesired non-specific T cell activation in specific immunotherapy. This is expected to result in a low risk of side effects.

[0136] Lysis of redirected target cells via recruitment of T cells with multispecific, at least bispecific, antibody constructs involves the formation of a cytolytic synapse and delivery of perforin and granzymes. Bound T cells are capable of continuous target cell lysis and are not subject to immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation (see, e.g., WO 2007 / 042261).

[0137] The cytotoxicity mediated by the antibody constructs of the invention can be measured in various ways. Effector cells can be, for example, stimulated enriched (human) CD8 positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are of macaque origin or express or are transfected with macaque MUC17 bound by the first domain, the effector cells should also be of macaque origin, such as a macaque T cell line, e.g. 4119LnPx. The target cells should express MUC17, e.g. human or macaque MUC17 (at least the extracellular domain of). The target cells can be a cell line (such as CHO) that is stably or transiently transfected with MUC17, e.g. human or macaque MUC17. Usually, EC 50 Values ​​are expected to be lower in target cell lines expressing high levels of MUC17 on the cell surface. The effector to target cell (E:T) ratio is usually about 10:1, but this can be varied. The cytotoxic activity of MUC17 bispecific antibody constructs is 51 It can be measured in a Cr release assay (incubation time of about 18 hours) or a cytotoxicity assay using FACS (incubation time of about 48 hours). The incubation time (cytotoxicity response) of the assay can be changed. Other methods for measuring cytotoxicity are well known to those skilled in the art and include MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonogenic assays and ECIS techniques.

[0138] The cytotoxic activity mediated by the MUC17xCD3 bispecific antibody construct of the invention is preferably measured in a cell-based cytotoxicity assay. 51 Cytotoxicity can also be measured by EC 50 The EC50 value corresponds to the half maximal effective concentration (the concentration of the antibody construct that induces a cytotoxic response halfway between the baseline and maximum). Preferably, the EC50 value for the MUC17xCD3 bispecific antibody construct is 50 The values ​​are ≦5000 pM or ≦4000 pM, more preferably ≦3000 pM or ≦2000 pM, even more preferably ≦1000 pM or ≦500 pM, even more preferably ≦400 pM or ≦300 pM, even more preferably ≦200 pM, even more preferably ≦100 pM, even more preferably ≦50 pM, even more preferably ≦20 pM or ≦10 pM and most preferably ≦5 pM.

[0139] In various assays, the EC given above 50 Values ​​can be measured. Stimulated / enriched CD8 + When T cells were used as effector cells, ECs were significantly higher than unstimulated PBMCs. 50 Those skilled in the art will recognize that the value can be expected to be low. 50 Values ​​can be expected to be lower if target cells express a large number of MUC17 compared to rats with fewer target expressors. For example, stimulated / enriched human CD8 + When T cells are used as effector cells (and either MUC17-transfected cells such as CHO cells or MUC17-positive human cell lines are used as target cells), the EC of the MUC17xCD3 bispecific antibody construct 50 The value is preferably ≦1000 pM, more preferably ≦500 pM, even more preferably ≦250 pM, even more preferably ≦100 pM, even more preferably ≦50 pM, even more preferably ≦10 pM and most preferably ≦5 pM. EC of MUC17×CD3 bispecific antibody construct, if used 50 The value is preferably ≦5000 pM or ≦4000 pM (particularly where the target cell is a MUC17 positive human cell line), more preferably ≦2000 pM (particularly where the target cell is a MUC17 transfected cell such as a CHO cell), more preferably ≦1000 pM or ≦500 pM, even more preferably ≦200 pM, even more preferably ≦150 pM, even more preferably ≦100 pM and most preferably ≦50 pM or less. When a macaque T cell line such as LnPx4119 is used as the effector cell and a macaque MUC17 transfected cell line such as a CHO cell is used as the target cell line, the EC 50 The value is preferably ≦2000 pM or ≦1500 pM, more preferably ≦1000 pM or ≦500 pM, even more preferably ≦300 pM or ≦250 pM, even more preferably ≦100 pM and most preferably ≦50 pM.

[0140] Preferably, the MUC17xCD3 bispecific antibody construct of the invention does not induce / mediate lysis or essentially does not induce / mediate lysis of MUC17-negative cells, such as CHO cells. The terms "does not induce lysis", "essentially does not induce lysis", "does not mediate lysis" or "essentially does not mediate lysis" mean that the antibody construct of the invention does not induce or mediate lysis of more than 30%, preferably more than 20%, more preferably more than 10%, particularly preferably more than 9%, 8%, 7%, 6% or 5% of MUC17-negative cells, taking the lysis of a MUC17-positive human cell line as 100%. This is usually the case for antibody construct concentrations up to 500 nM. The skilled person knows how to measure cell lysis without further effort. Moreover, specific instructions on how to measure cell lysis are taught herein.

[0141] The difference in cytotoxic activity between the monomeric and dimeric isoforms of a particular MUC17xCD3 bispecific antibody construct is referred to as the "potency gap." This potency gap can be measured, for example, by comparing the EC 50 Values ​​and EC of dimeric form 50 The potency gap of the MUC17xCD3 bispecific antibody constructs of the invention is preferably ≦5, more preferably ≦4, even more preferably ≦3, even more preferably ≦2 and most preferably ≦1.

[0142] The first and / or second (or any further) binding domains of the antibody construct of the invention are preferably cross-species specific in members of the mammalian order of primates. Cross-species specific CD3 binding domains are described, for example, in WO 2008 / 119567. According to one embodiment, the first and / or second binding domain, in addition to binding to human MUC17 and human CD3, will also bind to MUC17 / CD3, respectively, of primates including, but not limited to, New World primates (such as marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus Oedipus) or squirrel monkeys (Saimiri sciureus)), Old World primates (such as baboons and macaques), gibbons and non-human hominins.

[0143] In one embodiment of the antibody construct of the invention, the first domain binds human MUC17 and further binds macaque MUC17, such as MUC17 of cynomolgus monkeys (Macaca fascicularis), more preferably binds macaque MUC17 expressed on the surface of a cell, such as, for example, a CHO or 293 cell. The affinity of the first domain for MUC17, preferably human MUC17, is preferably ≦100 nM or ≦50 nM, more preferably ≦25 nM or ≦20 nM, more preferably ≦15 nM or ≦10 nM, even more preferably ≦5 nM, even more preferably ≦2.5 M or ≦2 M, even more preferably ≦1 nM, even more preferably ≦0.6 nM, even more preferably ≦0.5 nM, and most preferably ≦0.4 nM. The affinity can be determined, for example, by BIAc The affinity of the first domain to macaque MUC17 can be measured in a β- or β-reactive protein assay or a Scatchard assay. Other methods of determining affinity are well known to those skilled in the art. The affinity of the first domain to macaque MUC17 is preferably ≦15 nM, more preferably ≦10 nM, even more preferably ≦5 nM, even more preferably ≦1 nM, even more preferably ≦0.5 nM, even more preferably ≦0.1 nM, and most preferably ≦0.05 nM or even ≦0.01 nM.

[0144] Preferably, the affinity gap for binding of an antibody construct according to the invention to macaque MUC17 to human MUC17 [ma MUC17:hu MUC17] (e.g. as determined by BiaCore or Scatchard analysis) is <100, preferably <20, more preferably <15, even more preferably <10, even more preferably <8, more preferably <6 and most preferably <2. A preferred range for the affinity gap for binding of an antibody construct according to the invention to macaque MUC17 to human MUC17 is 0.1-20, more preferably 0.2-10, even more preferably 0.3-6, even more preferably 0.5-3 or 0.5-2.5 and most preferably 0.5-2 or 0.6-2.

[0145] The second domain of the antibody construct of the invention binds to human CD3 epsilon and / or macaque CD3 epsilon. In a preferred embodiment, the second domain further binds to Callithrix jacchus, Saguinus Oedipus or Saimiri sciureus CD3 epsilon. Both Callithrix jacchus and Saguinus oedipus are New World primates belonging to the family Callitrichidae, while Saimiri sciureus is a New World primate belonging to the family Cebidae.

[0146] With respect to the antibody construct of the invention, the second binding domain which binds to an extracellular epitope of the human and / or macaque CD3 epsilon chain comprises: (a) CDR-L1 as set forth in SEQ ID NO: 27 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 28 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 29 of WO 2008 / 119567; (b) CDR-L1 as set forth in SEQ ID NO: 117 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 118 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 119 of WO 2008 / 119567; and (c) CDR-L1 as set forth in SEQ ID NO: 153 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 154 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 155 of WO 2008 / 119567. It is preferred that the VL region comprises CDR-L1, CDR-L2 and CDR-L3 selected from:

[0147] In a further preferred embodiment of the antibody construct of the invention, the second domain binding to an extracellular epitope of the human and / or macaque CD3 epsilon chain comprises (a) CDR-H1 as set forth in SEQ ID NO: 12 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 13 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 14 of WO 2008 / 119567; (b) CDR-H1 as set forth in SEQ ID NO: 30 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 31 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 32 of WO 2008 / 119567; (c) CDR-H1 as set forth in SEQ ID NO: 48 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 49 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 50 of WO 2008 / 119567; (d) CDR-H1 as set forth in SEQ ID NO: 66 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 67 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 68 of WO 2008 / 119567; (e) CDR-H1 as set forth in SEQ ID NO: 84 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 85 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 86 of WO 2008 / 119567; (f) CDR-H1 as set forth in SEQ ID NO: 102 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 103 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 104 of WO 2008 / 119567; (g) CDR-H1 as set forth in SEQ ID NO: 120 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 121 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 122 of WO 2008 / 119567; (h) CDR-H1 as set forth in SEQ ID NO: 138 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 139 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 140 of WO 2008 / 119567; (i) CDR-H1 as set forth in SEQ ID NO: 156 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 157 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 158 of WO 2008 / 119567; and (j) CDR-H1 as set forth in SEQ ID NO: 174 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 175 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 176 of WO 2008 / 119567. The VH region comprises CDR-H1, CDR-H2 and CDR-H3 selected from:

[0148] In a preferred embodiment of the antibody construct of the invention, the above three sets of VL CDRs are combined with the above ten sets of VH CDRs in the second binding domain to form a set (30) comprising CDR-L1-3 and CDR-H1-3, respectively.

[0149] With regard to the antibody construct of the present invention, it is preferred that the second domain that binds to CD3 comprises a VL region selected from the group consisting of those set out in SEQ ID NO: 17, 21, 35, 39, 53, 57, 71, 75, 89, 93, 107, 111, 125, 129, 143, 147, 161, 165, 179 or 183 of WO2008 / 119567 or set out in SEQ ID NO: 13 according to the present invention.

[0150] The second domain that binds to CD3 is selected from the group consisting of SEQ ID NOs: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 48 It is also preferred that the VH region comprises a VH region selected from the group consisting of: 109, 123, 127, 141, 145, 159, 163, 177 or 181 or as set forth in SEQ ID NO:14.

[0151] More preferably, the antibody construct of the invention comprises: (a) a VL region as set forth in SEQ ID NO: 17 or 21 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 15 or 19 of WO 2008 / 119567; (b) the VL region as set forth in SEQ ID NO: 35 or 39 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 33 or 37 of WO 2008 / 119567; (c) the VL region as set forth in SEQ ID NO: 53 or 57 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 51 or 55 of WO 2008 / 119567; (d) the VL region as set forth in SEQ ID NO: 71 or 75 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 69 or 73 of WO 2008 / 119567; (e) the VL region as set forth in SEQ ID NO: 89 or 93 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 87 or 91 of WO 2008 / 119567; (f) the VL region as set forth in SEQ ID NO: 107 or 111 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 105 or 109 of WO 2008 / 119567; (g) the VL region as set forth in SEQ ID NO: 125 or 129 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 123 or 127 of WO 2008 / 119567; (h) the VL region as set forth in SEQ ID NO: 143 or 147 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 141 or 145 of WO 2008 / 119567; (i) the VL region as set forth in SEQ ID NO: 161 or 165 of WO 2008 / 119567 and the VH region as set forth in SEQ ID NO: 159 or 163 of WO 2008 / 119567; and (j) the VL region shown in SEQ ID NO: 179 or 183 of WO 2008 / 119567 and the VH region shown in SEQ ID NO: 177 or 181 of WO 2008 / 119567 and a second domain that binds to DC3, the second domain comprising a VL region and a VH region selected from the group consisting of:

[0152] Also preferred in the context of an antibody construct of the invention is a second domain that binds to CD3 comprising a VL region as shown in SEQ ID NO:13 and a VH region as shown in SEQ ID NO:14.

[0153] According to a preferred embodiment of the antibody construct of the invention, the first and / or second domain has the following format: the pair of VH and VL domains is in the format of a single chain antibody (scFv). The VH and VL domains are arranged in the order VH-VL or VL-VH. It is preferred that the VH domain is arranged at the N-terminus of the linker sequence and the VL domain is arranged at the C-terminus of the linker sequence.

[0154] Preferred embodiments of the above antibody constructs of the present invention are those described in SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, and a CD3-binding second domain comprising an amino acid sequence selected from the group consisting of 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187, or the amino acid sequence set forth in SEQ ID NO:15.

[0155] The first binding domain of the antibody construct of the present invention comprises: (a) CDR-L1 as set forth in SEQ ID NO: 36, CDR-L2 as set forth in SEQ ID NO: 37, and CDR-L3 as set forth in SEQ ID NO: 38, and CDR-H1 as set forth in SEQ ID NO: 33, CDR-H2 as set forth in SEQ ID NO: 34, and CDR-H3 as set forth in SEQ ID NO: 35; (b) CDR-L1 as set forth in SEQ ID NO: 47, CDR-L2 as set forth in SEQ ID NO: 48, and CDR-L3 as set forth in SEQ ID NO: 49, and CDR-H1 as set forth in SEQ ID NO: 44, CDR-H2 as set forth in SEQ ID NO: 45, and CDR-H3 as set forth in SEQ ID NO: 46; (c) CDR-L1 as set forth in SEQ ID NO: 58, CDR-L2 as set forth in SEQ ID NO: 59, and CDR-L3 as set forth in SEQ ID NO: 60, and CDR-H1 as set forth in SEQ ID NO: 55, CDR-H2 as set forth in SEQ ID NO: 56, and CDR-H3 as set forth in SEQ ID NO: 57; (d) CDR-L1 as set forth in SEQ ID NO: 69, CDR-L2 as set forth in SEQ ID NO: 70, and CDR-L3 as set forth in SEQ ID NO: 71, and CDR-H1 as set forth in SEQ ID NO: 66, CDR-H2 as set forth in SEQ ID NO: 67, and CDR-H3 as set forth in SEQ ID NO: 68; (e) CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, and CDR-L3 as set forth in SEQ ID NO: 82, and CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 78, and CDR-H3 as set forth in SEQ ID NO: 79; (f) CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93, and CDR-H1 as set forth in SEQ ID NO: 88, CDR-H2 as set forth in SEQ ID NO: 89, and CDR-H3 as set forth in SEQ ID NO: 90; (g) CDR-L1 as set forth in SEQ ID NO: 102, CDR-L2 as set forth in SEQ ID NO: 103, and CDR-L3 as set forth in SEQ ID NO: 104, and CDR-H1 as set forth in SEQ ID NO: 99, CDR-H2 as set forth in SEQ ID NO: 100, and CDR-H3 as set forth in SEQ ID NO: 101; (h) CDR-L1 as set forth in SEQ ID NO: 113, CDR-L2 as set forth in SEQ ID NO: 114, and CDR-L3 as set forth in SEQ ID NO: 115, and CDR-H1 as set forth in SEQ ID NO: 110, CDR-H2 as set forth in SEQ ID NO: 111, and CDR-H3 as set forth in SEQ ID NO: 112; (i) CDR-L1 as set forth in SEQ ID NO: 124, CDR-L2 as set forth in SEQ ID NO: 125, and CDR-L3 as set forth in SEQ ID NO: 126, and CDR-H1 as set forth in SEQ ID NO: 121, CDR-H2 as set forth in SEQ ID NO: 122, and CDR-H3 as set forth in SEQ ID NO: 123; (j) CDR-L1 as set forth in SEQ ID NO: 135, CDR-L2 as set forth in SEQ ID NO: 136, and CDR-L3 as set forth in SEQ ID NO: 137, and CDR-H1 as set forth in SEQ ID NO: 132, CDR-H2 as set forth in SEQ ID NO: 133, and CDR-H3 as set forth in SEQ ID NO: 134; (k) CDR-L1 as set forth in SEQ ID NO: 146, CDR-L2 as set forth in SEQ ID NO: 147, and CDR-L3 as set forth in SEQ ID NO: 148, and CDR-H1 as set forth in SEQ ID NO: 143, CDR-H2 as set forth in SEQ ID NO: 144, and CDR-H3 as set forth in SEQ ID NO: 145; (l) CDR-L1 as set forth in SEQ ID NO: 157, CDR-L2 as set forth in SEQ ID NO: 159 and CDR-H1 as set forth in SEQ ID NO: 154, CDR-H2 as set forth in SEQ ID NO: 155 and CDR-H3 as set forth in SEQ ID NO: 156; (m) CDR-L1 as set forth in SEQ ID NO: 168, CDR-L2 as set forth in SEQ ID NO: 169, and CDR-L3 as set forth in SEQ ID NO: 170, and CDR-H1 as set forth in SEQ ID NO: 165, CDR-H2 as set forth in SEQ ID NO: 166, and CDR-H3 as set forth in SEQ ID NO: 167; (n) CDR-L1 as set forth in SEQ ID NO: 179, CDR-L2 as set forth in SEQ ID NO: 180, and CDR-L3 as set forth in SEQ ID NO: 181, and CDR-H1 as set forth in SEQ ID NO: 176, CDR-H2 as set forth in SEQ ID NO: 177, and CDR-H3 as set forth in SEQ ID NO: 178; (o) CDR-L1 as set forth in SEQ ID NO: 190, CDR-L2 as set forth in SEQ ID NO: 191, and CDR-L3 as set forth in SEQ ID NO: 192, and CDR-H1 as set forth in SEQ ID NO: 187, CDR-H2 as set forth in SEQ ID NO: 188, and CDR-H3 as set forth in SEQ ID NO: 189; (p) CDR-L1 as set forth in SEQ ID NO: 201, CDR-L2 as set forth in SEQ ID NO: 202, and CDR-L3 as set forth in SEQ ID NO: 203, and CDR-H1 as set forth in SEQ ID NO: 198, CDR-H2 as set forth in SEQ ID NO: 199, and CDR-H3 as set forth in SEQ ID NO: 200; (q) CDR-L1 as set forth in SEQ ID NO: 212, CDR-L2 as set forth in SEQ ID NO: 213, and CDR-L3 as set forth in SEQ ID NO: 214, and CDR-H1 as set forth in SEQ ID NO: 209, CDR-H2 as set forth in SEQ ID NO: 210, and CDR-H3 as set forth in SEQ ID NO: 211; (r) CDR-L1 as set forth in SEQ ID NO: 223, CDR-L2 as set forth in SEQ ID NO: 224, and CDR-L3 as set forth in SEQ ID NO: 225, and CDR-H1 as set forth in SEQ ID NO: 220, CDR-H2 as set forth in SEQ ID NO: 221, and CDR-H3 as set forth in SEQ ID NO: 222; (s) CDR-L1 as set forth in SEQ ID NO: 234, CDR-L2 as set forth in SEQ ID NO: 235, and CDR-L3 as set forth in SEQ ID NO: 236, and CDR-H1 as set forth in SEQ ID NO: 231, CDR-H2 as set forth in SEQ ID NO: 232, and CDR-H3 as set forth in SEQ ID NO: 233; (t) CDR-L1 as set forth in SEQ ID NO: 245, CDR-L2 as set forth in SEQ ID NO: 246, and CDR-L3 as set forth in SEQ ID NO: 247, and CDR-H1 as set forth in SEQ ID NO: 242, CDR-H2 as set forth in SEQ ID NO: 243, and CDR-H3 as set forth in SEQ ID NO: 244; (u) CDR-L1 as set forth in SEQ ID NO: 256, CDR-L2 as set forth in SEQ ID NO: 257, and CDR-L3 as set forth in SEQ ID NO: 258, and CDR-H1 as set forth in SEQ ID NO: 253, CDR-H2 as set forth in SEQ ID NO: 254, and CDR-H3 as set forth in SEQ ID NO: 255; (v) CDR-L1 as set forth in SEQ ID NO:267, CDR-L2 as set forth in SEQ ID NO:268, and CDR-L3 as set forth in SEQ ID NO:269, and CDR-H1 as set forth in SEQ ID NO:264, CDR-H2 as set forth in SEQ ID NO:265, and CDR-H3 as set forth in SEQ ID NO:266; (w) CDR-L1 as set forth in SEQ ID NO: 278, CDR-L2 as set forth in SEQ ID NO: 279, and CDR-L3 as set forth in SEQ ID NO: 280, and CDR-H1 as set forth in SEQ ID NO: 275, CDR-H2 as set forth in SEQ ID NO: 276, and CDR-H3 as set forth in SEQ ID NO: 276; (x) CDR-L1 as set forth in SEQ ID NO: 289, CDR-L2 as set forth in SEQ ID NO: 290, and CDR-L3 as set forth in SEQ ID NO: 291, and CDR-H1 as set forth in SEQ ID NO: 286, and CDR-H2 as set forth in SEQ ID NO: 287 and CDR-H3 as set forth in SEQ ID NO:288; (y) CDR-L1 as set forth in SEQ ID NO: 300, CDR-L2 as set forth in SEQ ID NO: 301, and CDR-L3 as set forth in SEQ ID NO: 302, and CDR-H1 as set forth in SEQ ID NO: 297, CDR-H2 as set forth in SEQ ID NO: 298, and CDR-H3 as set forth in SEQ ID NO: 299; (z) CDR-L1 as set forth in SEQ ID NO: 311, CDR-L2 as set forth in SEQ ID NO: 312, and CDR-L3 as set forth in SEQ ID NO: 313, and CDR-H1 as set forth in SEQ ID NO: 308, CDR-H2 as set forth in SEQ ID NO: 309, and CDR-H3 as set forth in SEQ ID NO: 310; (aa) CDR-L1 as set forth in SEQ ID NO: 322, CDR-L2 as set forth in SEQ ID NO: 323, and CDR-L3 as set forth in SEQ ID NO: 324, and CDR-H1 as set forth in SEQ ID NO: 319, CDR-H2 as set forth in SEQ ID NO: 320, and CDR-H3 as set forth in SEQ ID NO: 321; (ab) CDR-L1 as set forth in SEQ ID NO: 333, CDR-L2 as set forth in SEQ ID NO: 334, and CDR-L3 as set forth in SEQ ID NO: 335, and CDR-H1 as set forth in SEQ ID NO: 330, CDR-H2 as set forth in SEQ ID NO: 331, and CDR-H3 as set forth in SEQ ID NO: 332; (ac) CDR-L1 as set forth in SEQ ID NO: 344, CDR-L2 as set forth in SEQ ID NO: 345, and CDR-L3 as set forth in SEQ ID NO: 346, and CDR-H1 as set forth in SEQ ID NO: 341, CDR-H2 as set forth in SEQ ID NO: 342, and CDR-H3 as set forth in SEQ ID NO: 343; (ad) CDR-L1 as set forth in SEQ ID NO: 355, CDR-L2 as set forth in SEQ ID NO: 356, and CDR-L3 as set forth in SEQ ID NO: 357, and CDR-H1 as set forth in SEQ ID NO: 352, CDR-H2 as set forth in SEQ ID NO: 353, and CDR-H3 as set forth in SEQ ID NO: 354; (ae) CDR-L1 as set forth in SEQ ID NO: 366, CDR-L2 as set forth in SEQ ID NO: 367, and CDR-L3 as set forth in SEQ ID NO: 368, and CDR-H1 as set forth in SEQ ID NO: 363, CDR-H2 as set forth in SEQ ID NO: 364, and CDR-H3 as set forth in SEQ ID NO: 365; (af) CDR-L1 as set forth in SEQ ID NO: 377, CDR-L2 as set forth in SEQ ID NO: 378, and CDR-L3 as set forth in SEQ ID NO: 379, and CDR-H1 as set forth in SEQ ID NO: 374, CDR-H2 as set forth in SEQ ID NO: 375, and CDR-H3 as set forth in SEQ ID NO: 376; (ag) CDR-L1 as set forth in SEQ ID NO: 388, CDR-L2 as set forth in SEQ ID NO: 389 and CDR-L3 as set forth in SEQ ID NO: 390, and CDR-H1 as set forth in SEQ ID NO: 385, CDR-H2 as set forth in SEQ ID NO: 386 and CDR-H3 as set forth in SEQ ID NO: 386; (ah) CDR-L1 as set forth in SEQ ID NO: 399, CDR-L2 as set forth in SEQ ID NO: 400, and CDR-L3 as set forth in SEQ ID NO: 401, and CDR-H1 as set forth in SEQ ID NO: 396, CDR-H2 as set forth in SEQ ID NO: 397, and CDR-H3 as set forth in SEQ ID NO: 398; (ai) CDR-L1 as set forth in SEQ ID NO: 410, CDR-L2 as set forth in SEQ ID NO: 411, and CDR-L3 as set forth in SEQ ID NO: 412, and CDR-H1 as set forth in SEQ ID NO: 407, CDR-H2 as set forth in SEQ ID NO: 408, and CDR-H3 as set forth in SEQ ID NO: 409; (aj) CDR-L1 as set forth in SEQ ID NO: 421, CDR-L2 as set forth in SEQ ID NO: 422, and CDR-L3 as set forth in SEQ ID NO: 423, and CDR-H1 as set forth in SEQ ID NO: 418, CDR-H2 as set forth in SEQ ID NO: 419, and CDR-H3 as set forth in SEQ ID NO: 420; (ak) CDR-L1 as set forth in SEQ ID NO: 432, CDR-L2 as set forth in SEQ ID NO: 435 and CDR-L3 as set forth in SEQ ID NO: 434, and CDR-H1 as set forth in SEQ ID NO: 429, CDR-H2 as set forth in SEQ ID NO: 430 and CDR-H3 as set forth in SEQ ID NO: 431; (a1) CDR-L1 as set forth in SEQ ID NO: 443, CDR-L2 as set forth in SEQ ID NO: 444, and CDR-L3 as set forth in SEQ ID NO: 445, and CDR-H1 as set forth in SEQ ID NO: 440, CDR-H2 as set forth in SEQ ID NO: 441, and CDR-H3 as set forth in SEQ ID NO: 442; (am) CDR-L1 as set forth in SEQ ID NO: 454, CDR-L2 as set forth in SEQ ID NO: 455, and CDR-L3 as set forth in SEQ ID NO: 456, and CDR-H1 as set forth in SEQ ID NO: 451, CDR-H2 as set forth in SEQ ID NO: 452, and CDR-H3 as set forth in SEQ ID NO: 453; (an) CDR-L1 as set forth in SEQ ID NO: 465, CDR-L2 as set forth in SEQ ID NO: 466, and CDR-L3 as set forth in SEQ ID NO: 467, and CDR-H1 as set forth in SEQ ID NO: 462, CDR-H2 as set forth in SEQ ID NO: 463, and CDR-H3 as set forth in SEQ ID NO: 464; (ao) CDR-L1 as set forth in SEQ ID NO: 476, CDR-L2 as set forth in SEQ ID NO: 477, and CDR-L3 as set forth in SEQ ID NO: 478, and CDR-H1 as set forth in SEQ ID NO: 473, CDR-H2 as set forth in SEQ ID NO: 474, and CDR-H3 as set forth in SEQ ID NO: 475; (ap) CDR-L1 as set forth in SEQ ID NO: 487, CDR-L2 as set forth in SEQ ID NO: 488 and CDR-L3 as set forth in SEQ ID NO: 489, and CDR-H1 as set forth in SEQ ID NO: 484, CDR-H2 as set forth in SEQ ID NO: 485 and CDR-H3 as set forth in SEQ ID NO: 486; (aq) CDR-L1 as set forth in SEQ ID NO: 498, CDR-L2 as set forth in SEQ ID NO: 499, and CDR-L3 as set forth in SEQ ID NO: 500, and CDR-H1 as set forth in SEQ ID NO: 495, CDR-H2 as set forth in SEQ ID NO: 496, and CDR-H3 as set forth in SEQ ID NO: 497; (ar) CDR-L1 as set forth in SEQ ID NO: 509, CDR-L2 as set forth in SEQ ID NO: 510 and CDR-L3 as set forth in SEQ ID NO: 511, and CDR-H1 as set forth in SEQ ID NO: 506, CDR-H2 as set forth in SEQ ID NO: 507 and CDR-H3 as set forth in SEQ ID NO: 508; and (as) CDR-L1 as set forth in SEQ ID NO: 520, CDR-L2 as set forth in SEQ ID NO: 521, and CDR-L3 as set forth in SEQ ID NO: 522, and CDR-H1 as set forth in SEQ ID NO: 517, CDR-H2 as set forth in SEQ ID NO: 518, and CDR-H3 as set forth in SEQ ID NO: 519. a VL region comprising CDR-L1, CDR-L2 and CDR-L3, and a VH region comprising CDR-H1, CDR-H2 and CDR-H3, selected from the group consisting of: (c) CDR-L1 as set forth in SEQ ID NO: 58, CDR-L2 as set forth in SEQ ID NO: 59, and CDR-L3 as set forth in SEQ ID NO: 60, and CDR-H1 as set forth in SEQ ID NO: 55, CDR-H2 as set forth in SEQ ID NO: 56, and CDR-H3 as set forth in SEQ ID NO: 57; (n) CDR-L1 as set forth in SEQ ID NO: 179, CDR-L2 as set forth in SEQ ID NO: 180, and CDR-L3 as set forth in SEQ ID NO: 181, and CDR-H1 as set forth in SEQ ID NO: 176, CDR-H2 as set forth in SEQ ID NO: 177, and CDR-H3 as set forth in SEQ ID NO: 178; (ac) CDR-L1 as set forth in SEQ ID NO: 344, CDR-L2 as set forth in SEQ ID NO: 345 and CDR-L3 as set forth in SEQ ID NO: 346, and CDR-H1 as set forth in SEQ ID NO: 341, CDR-H2 as set forth in SEQ ID NO: 342 and CDR-H3 as set forth in SEQ ID NO: 343; and (aj) CDR-L1 as set forth in SEQ ID NO: 421, CDR-L2 as set forth in SEQ ID NO: 423 and CDR-H1 as set forth in SEQ ID NO: 418, CDR-H2 as set forth in SEQ ID NO: 419 and CDR-H3 as set forth in SEQ ID NO: 420 It is also envisaged that is preferred.

[0156] The first binding domain of the antibody construct of the present invention comprises: (a) a VL region as set forth in SEQ ID NO: 40 and a VH region as set forth in SEQ ID NO: 39; (b) a VL region as set forth in SEQ ID NO:51 and a VH region as set forth in SEQ ID NO:50; (c) a VL region as set forth in SEQ ID NO:62 and a VH region as set forth in SEQ ID NO:61; (d) a VL region as set forth in SEQ ID NO: 73 and a VH region as set forth in SEQ ID NO: 72; (e) a VL region as set forth in SEQ ID NO: 84 and a VH region as set forth in SEQ ID NO: 83; (f) a VL region as set forth in SEQ ID NO: 95 and a VH region as set forth in SEQ ID NO: 94; (g) a VL region as set forth in SEQ ID NO: 106 and a VH region as set forth in SEQ ID NO: 105; (h) a VL region as set forth in SEQ ID NO: 117 and a VH region as set forth in SEQ ID NO: 116; (i) a VL region as set forth in SEQ ID NO: 128 and a VH region as set forth in SEQ ID NO: 127; (j) a VL region as set forth in SEQ ID NO: 139 and a VH region as set forth in SEQ ID NO: 138; (k) a VL region as set forth in SEQ ID NO: 150 and a VH region as set forth in SEQ ID NO: 149; (l) a VL region as set forth in SEQ ID NO: 161 and a VH region as set forth in SEQ ID NO: 160; (m) a VL region as set forth in SEQ ID NO: 172 and a VH region as set forth in SEQ ID NO: 171; (n) a VL region as set forth in SEQ ID NO: 183 and a VH region as set forth in SEQ ID NO: 182; (o) a VL region as set forth in SEQ ID NO: 194 and a VH region as set forth in SEQ ID NO: 193; (p) a VL region as set forth in SEQ ID NO: 205 and a VH region as set forth in SEQ ID NO: 204; (q) a VL region as set forth in SEQ ID NO: 216 and a VH region as set forth in SEQ ID NO: 215; (r) a VL region as set forth in SEQ ID NO: 227 and a VH region as set forth in SEQ ID NO: 226; (s) a VL region as set forth in SEQ ID NO: 238 and a VH region as set forth in SEQ ID NO: 237; (t) a VL region as set forth in SEQ ID NO: 249 and a VH region as set forth in SEQ ID NO: 248; (u) a VL region as set forth in SEQ ID NO: 260 and a VH region as set forth in SEQ ID NO: 259; (v) a VL region as set forth in SEQ ID NO: 271 and a VH region as set forth in SEQ ID NO: 270; (w) a VL region as set forth in SEQ ID NO: 282 and a VH region as set forth in SEQ ID NO: 281; (x) a VL region as set forth in SEQ ID NO: 293 and a VH region as set forth in SEQ ID NO: 292; (y) a VL region as set forth in SEQ ID NO: 304 and a VH region as set forth in SEQ ID NO: 303; (z) a VL region as set forth in SEQ ID NO: 315 and a VH region as set forth in SEQ ID NO: 314; (aa) a VL region as set forth in SEQ ID NO: 326 and a VH region as set forth in SEQ ID NO: 325; (ab) a VL region as set forth in SEQ ID NO: 337 and a VH region as set forth in SEQ ID NO: 336; (ac) a VL region as set forth in SEQ ID NO: 348 and a VH region as set forth in SEQ ID NO: 347; (ad) a VL region as set forth in SEQ ID NO: 359 and a VH region as set forth in SEQ ID NO: 358; (ae) a VL region as set forth in SEQ ID NO: 370 and a VH region as set forth in SEQ ID NO: 369; (af) a VL region as set forth in SEQ ID NO: 381 and a VH region as set forth in SEQ ID NO: 380; (ag) a VL region as set forth in SEQ ID NO: 392 and a VH region as set forth in SEQ ID NO: 391; (ah) a VL region as set forth in SEQ ID NO: 403 and a VH region as set forth in SEQ ID NO: 402; (ai) a VL region as set forth in SEQ ID NO: 414 and a VH region as set forth in SEQ ID NO: 413; (aj) a VL region as set forth in SEQ ID NO: 425 and a VH region as set forth in SEQ ID NO: 424; (ak) a VL region as set forth in SEQ ID NO: 436 and a VH region as set forth in SEQ ID NO: 435; (a1) a VL region as set forth in SEQ ID NO: 447 and a VH region as set forth in SEQ ID NO: 446; (am) a VL region as set forth in SEQ ID NO: 458 and a VH region as set forth in SEQ ID NO: 457; (an) a VL region as set forth in SEQ ID NO: 469 and a VH region as set forth in SEQ ID NO: 468; (ao) a VL region as set forth in SEQ ID NO: 480 and a VH region as set forth in SEQ ID NO: 479; (ap) a VL region as set forth in SEQ ID NO: 491 and a VH region as set forth in SEQ ID NO: 490; (aq) a VL region as set forth in SEQ ID NO: 502 and a VH region as set forth in SEQ ID NO: 501; (ar) a VL region as set forth in SEQ ID NO: 513 and a VH region as set forth in SEQ ID NO: 512; and (as) a VL region as set forth in SEQ ID NO: 524 and a VH region as set forth in SEQ ID NO: 523 It is further envisaged that the antibody comprises a VH region and a VL region selected from the group consisting of:

[0157] The first binding domain of the antibody construct of the present invention is selected from the group consisting of SEQ ID NOs: 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 184, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514 and 525 or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity thereto.

[0158] The present invention relates to the nucleic acids represented by SEQ ID NOs: 42, 43, 53, 54, 64, 65, 75, 76, 86, 87, 97, 98, 108, 109, 119, 120, 130, 131, 141, 142, 152, 153, 163, 164, 174, 175, 185, 186, 196, 197, 207, 208, 218, 219, 229, 230, 240, 241, 251, 252, 262, 263, 273, 274, 284, 285, 295, 296, 306, 307, 317, 318, 328, 329, 339, 340, 350, 351, 361, 362, 372, 374, 376, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 412, 413, 414, 415, 416, 4

[0033] Further provided is an antibody construct comprising or having an amino acid sequence selected from the group consisting of: 73, 383, 384, 394, 395, 405, 406, 416, 417, 427, 428, 438, 439, 449, 450, 460, 461, 471, 472, 482, 483, 493, 494, 504, 505, 515, 516, 526 and 527 or having an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity thereto (fully bispecific antibody constructs).

[0159] Covalent modifications of antibody constructs are also included within the scope of the present invention, which are usually, but not always, performed post-translationally. For example, some types of covalent modifications of antibody constructs are introduced into the molecule by reacting specific amino acid residues of the antibody construct with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0160] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives.Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0161] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, and the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydrides; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0162] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues can be accomplished by the reaction: must be carried out under alkaline conditions. Moreover, these reagents can react with lysine groups and with the arginine epsilon-amino group.

[0163] The specific modification of tyrosyl residues may be undertaken for the purpose of introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 The chloramine T method described above, in which tyrosyl residues are iodinated with I to prepare labeled proteins for use in radioimmunoassay, is preferred.

[0164] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N-R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0165] Derivatization with bifunctional agents is useful for crosslinking the antibody constructs of the present invention to water-insoluble support matrices or surfaces for use in a variety of methods. Commonly used crosslinking agents include, for example, homobifunctional imidoesters including 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate provide photoactivatable intermediates capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are used for protein immobilization.

[0166] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues is included within the scope of the invention.

[0167] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0168] Another type of covalent modification of an antibody construct included within the scope of the invention involves altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below) or the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0169] Glycosylation of polypeptides is usually either N-linked or O-linked. O-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the addition of a single sugar, N-acetylgalactosamine, galactose or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0170] Glycosylation sites may be conveniently added to the antibody construct by modifying the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). Modification may also be made by the addition to or substitution by one or more serine or threonine residues from the start sequence (for O-linked glycosylation sites). Briefly, it is preferred to modify the amino acid sequence of the antibody construct by modification at the DNA level, in particular by mutating the DNA encoding the polypeptide at preselected bases to generate codons that will be translated into the desired amino acid.

[0171] Another means of increasing the number of carbohydrate moieties on an antibody construct is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N-linked and O-linked glycosylation. Depending on the mode of attachment used, sugars can be added to (a) arginines and histidines, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0172] Removal of carbohydrate moieties present on the starting antibody construct can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment cleaves most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides is described by Thotakura et al. This can be achieved by the use of various endo- and exo-glycosidases as described by Duskin et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

[0173] Other modifications of the antibody construct are also contemplated herein. For example, another type of covalent modification of the antibody construct includes linking the antibody construct to various non-proteinaceous polymers, including various polyols, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or combinations of polyethylene glycol and polypropylene glycol, in the manner set forth in U.S. Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antibody construct to facilitate the addition of polymers such as, for example, PEG.

[0174] In some embodiments, the covalent modification of the antibody construct of the present invention comprises the addition of one or more labels. To reduce potential steric hindrance, the labeling group can be attached to the antibody construct via a spacer arm of various lengths. A variety of methods for labeling proteins are known in the art and can be used in carrying out the present invention. The term "label" or "labeling group" refers to any detectable label. In general, labels are divided into various classes depending on the assay in which the label is to be detected, examples of which include, but are not limited to, the following: a) Radioisotopes or radionuclides (e.g. 3 H, 14 C. 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 I) Isotopic labels, which may be radioisotopes or heavy isotopes, such as b) Magnetic labels (e.g., magnetic particles) c) redox-active moieties d) optical dyes (including but not limited to chromophores, fluorophores and fluorophores) such as fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), chemiluminescent groups and fluorophores, which can be either "small molecule" fluorophores or proteinaceous fluorophores; e) Enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase) f) Biotinylation Group g) A predetermined polypeptide epitope recognized by a secondary reporter (eg, a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.).

[0175] "Fluorescent label" refers to any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Suitable optical dyes, including fluorophores, include, but are not limited to, Fluorescent Probes, Eugene, OR), FITC, rhodamine and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland.

[0176] Suitable proteinaceous fluorescent labels include GFP from Renilla, Ptilosarcus or Aequorea species (Chalfie et al., 1994, Science 263:802-805), green fluorescent protein including EGFP (Clontech Laboratories, Inc., Genbank Accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1999, Biochemistry 26:131-132; Heim et al., 1999, Biochemistry 26:131-132; Heim et al., 1999, Biochemistry 26:131-132). t al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al., 1997, J. Immunol. 150:5408-5417), and β-galactosidase (Nolan et al., 1998, J. Immunol. 150:5408-5417). al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607) and Renilla (WO 92 / 15673; WO 95 / 07463; WO 98 / 14605; WO 98 / 26277; WO 99 / 49019; U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558).

[0177] The antibody constructs of the present invention may also include additional domains, e.g., useful for the isolation of the molecule or relevant for adapting the pharmacokinetic profile of the molecule. The domains useful for the isolation of the antibody construct may be selected from peptide motifs or secondarily introduced moieties that can be captured by an isolation method, e.g., an isolation column. Non-limiting examples of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin binding domains (CBD tags), maltose binding protein (MBP tags), Flag tags, Strep tags and variants thereof (e.g., StrepII tags) and His tags. All of the antibody constructs disclosed herein may include a His tag domain, commonly known as a repeat of consecutive His residues, preferably five and more preferably six His residues (hexahistidine) in the amino acid sequence of the molecule. The His tag may, for example, be located at either the N-terminus or the C-terminus of the antibody construct, but is preferably located at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 16) is attached to the C-terminus of the antibody construct according to the present invention via a peptide bond. In addition, the conjugate system of PLGA-PEG-PLGA can be combined with a polyhistidine tag for sustained release applications and improved pharmacokinetic profile.

[0178] Amino acid sequence modifications of the antibody constructs described herein are also contemplated. For example, improving the binding affinity and / or other biological properties of the antibody construct may be desired. Amino acid sequence variants of the antibody construct are generated by introducing appropriate nucleotide changes into the nucleic acid of the antibody construct or by peptide synthesis. All of the amino acid sequence modifications described below should result in an antibody construct that still retains the desired biological activity (binding to MUC17 and CD3) of the unmodified parent molecule.

[0179] The term "amino acid" or "amino acid residue" generally refers to an amino acid having its art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as appropriate. In general, amino acids can be classified according to the presence of a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp and Tyr).

[0180] Amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody construct. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processes of the antibody construct, such as changing the number or position of glycosylation sites.

[0181] For example, 1, 2, 3, 4, 5 or 6 amino acids may be inserted, substituted or deleted in each of the CDRs (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be inserted, substituted or deleted in each of the FRs. Preferably, insertions of amino acid sequences into the antibody constructs include amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Corresponding modifications may be made within the third domain of the antibody constructs of the invention. Insertion variants of the antibody constructs of the invention include fusions of enzymes or fusions to polypeptides to the N-terminus or C-terminus of the antibody construct.

[0182] The most important sites for substitution mutagenesis include (but are not limited to) the CDRs of heavy and / or light chains, especially the hypervariable regions, but also contemplate modification of FRs in heavy and / or light chains. Substitutions are preferably conservative substitutions as described herein. Preferably, depending on the length of the CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids may be substituted in the CDRs, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be substituted in the framework regions (FRs). For example, if the CDR sequence comprises 6 amino acids, it is envisaged that 1, 2 or 3 of these amino acids are substituted. Similarly, if the CDR sequence comprises 15 amino acids, it is envisaged that 1, 2, 3, 4, 5 or 6 of these amino acids are substituted.

[0183] A useful method for identifying specific residues or regions of an antibody construct that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells in Science, 244:1081-1085 (1989). In this method, a residue or target residues within the antibody construct are identified (e.g., charged residues such as arg, asp, his, lys, and glu) that affect the interaction of the amino acid with the epitope, and are replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine).

[0184] Next, further or other variants are introduced at or in place of the substitution site to select those amino acid positions that show functional sensitivity to the substitution. Thus, the site or region where the amino acid sequence variant is introduced is predetermined, but the nature of the mutation itself need not be predetermined. For example, to analyze or optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be performed at the target codon or target region, and the variants of the expressed antibody construct are screened for the optimal combination of desired activity. Techniques for making substitution mutations at a predetermined site in DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using an assay for antigen binding activity, such as MUC17 or CD3 binding.

[0185] Generally, when amino acids are substituted in one or more or all of the CDRs of the heavy and / or light chains, the resulting "substituted" sequence will have at least a partial similarity to the "original" CDR sequence. Preferably, the CDR is at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85% and especially preferably 90% or 95% identical to the "replaced" sequence. This means that how identical it is to the "replaced" sequence depends on the length of the CDR. For example, a CDR with 5 amino acids is preferably 80% identical to its replaced sequence, since it has at least one replaced amino acid. Thus, the CDRs of an antibody construct may have different degrees of identity to their replaced sequences, for example CDRL1 may have 80%, while CDRL3 may have 90%.

[0186] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 3 below) is envisaged, as long as the antibody construct retains the ability to bind MUC17 via the first binding domain and CD3 epsilon via the second binding domain, and / or its CDRs have identity to the substituted sequences (at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85% and especially preferably 90% or 95% identical to the "original" CDR sequences).

[0187] Conservative substitutions are shown under the heading of "preferred substitutions" in Table 3. If such substitutions alter biological activity, more substantial changes, referred to in Table 3 as "exemplary substitutions," or described further below in connection with amino acid classes, can be introduced and the products screened for desired characteristics.

[0188] [Table 3]

[0189] Substantial alterations in the biological properties of the antibody constructs of the invention are achieved by selecting substitutions that differ significantly in their impact on (a) the structure of the polypeptide backbone in the substituted region, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are classified into the following groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0190] Non-conservative substitutions would entail exchanging a member of one of these classes for another. Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody construct may be substituted, generally with serine, to improve the oxidative stability of the molecule and avoid aberrant crosslinking. Conversely, the addition of cysteine ​​bond(s) to the antibody may improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0191] With respect to amino acid sequences, sequence identity and / or similarity can be determined using standard techniques known in the art, including, but not limited to, those described by Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by visual inspection. Preferably, the percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0192] One example of a useful algorithm is PILEUP. PILEUP generates a multiple sequence alignment from a group of related sequences using a progressive pairwise alignment method. It can also plot a tree showing the clustering relationships used to generate the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360. The method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0193] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap coverage=1, overlap percentage=0.125, word threshold (T)=II. The HSP S and HSP S2 parameters are dynamic values ​​and are set by the program itself depending on the composition of the individual sequence and the composition of the particular database against which the sequence of interest is being searched, but the values ​​may be adjusted to increase sensitivity.

[0194] Another useful algorithm is Gapped BLAST as reported in Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score, the threshold T parameter is set to 9, the two-hit method for producing ungapped extensions has a cost of 10+k for a gap length k, Xu is set to 16, and Xg is set to 40 for the database search stage and 67 for the output stage of the algorithm. Gapped alignments are produced with a score corresponding to approximately 22 bits.

[0195] Generally, the amino acid homology, similarity or identity between the individual variant CDR or VH / VL sequences is at least 60% relative to the sequences shown herein, and more typically, it is preferred that the homology or identity is increased to at least 65% or 70%, more preferably at least 75% or 80%, even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even near 100%. Similarly, "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequences of the binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of the antibody construct. A specific method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters with overlap range and overlap percentage of 1 and 0.125, respectively.

[0196] Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequence encoding each variant CDR or VH / VL sequence and the nucleotide sequences set out herein will be at least 60%, more typically the homology or identity will be at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and preferably increasing to nearly 100%. Thus, a "variant CDR" or "variant VH / VL region" is one that has a particular homology, similarity or identity to a parent CDR / VH / VL of the invention and shares a biological function, including but not limited to, at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR or VH / VL.

[0197] In one embodiment, the percentage of identity of the antibody construct according to the invention to the human germline is ≧70% or ≧75%, more preferably ≧80% or ≧85%, even more preferably ≧90% and most preferably ≧91%, ≧92%, ≧93%, ≧94%, ≧95% or even ≧96%. Identity to human antibody germline gene products is considered to be an important feature to reduce the risk of a therapeutic protein eliciting an immune response to the drug in patients under treatment. Hwang & Foote ("Immunogenicity of engineered antibodies"; Methods 36 (2005) 3-10) have demonstrated that reducing the non-human portion of a drug-antibody construct results in a reduced risk of eliciting anti-drug antibodies in patients under treatment. By comparing a large number of clinically evaluated antibody drugs and the corresponding immunogenicity data, humanization of the antibody V-regions shows a trend towards less immunogenicity of the protein (5.1% of patients on average) than antibodies carrying unmodified non-human V-regions (23.59% of patients on average). Therefore, for protein therapeutics in the form of V-region-based antibody constructs, a high identity to human sequences is desirable. To determine this germline identity, the V region of the VL can be aligned with the amino acid sequences of human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the percentage of amino acid sequence can be calculated by dividing the number of identical amino acid residues by the total number of amino acid residues in the VL. A similar method can be used for the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), with the exception that the VH CDR3 can be excluded due to its high diversity and lack of existing human germline VH CDR3 alignment partners. Recombinant techniques can then be used to increase the sequence identity to human antibody germline genes.

[0198] In a further embodiment, the bispecific antibody constructs of the invention exhibit high monomer yields under standard laboratory scale conditions, e.g., in a standard two-step purification process. Preferably, the monomer yield of the antibody construct according to the invention is ≧0.25 mg / L supernatant, more preferably ≧0.5 mg / L, even more preferably ≧1 mg / L and most preferably ≧3 mg / L supernatant.

[0199] Similarly, the yield of dimeric antibody construct isoforms of the antibody construct and therefore the monomer percentage (i.e. monomer:(monomer+dimer)) can be determined. The productivity of monomeric and dimeric antibody constructs and the calculated monomer percentage can be obtained, for example, by SEC purification step of culture supernatants from standardized research scale production in roller bottles. In one embodiment, the monomer percentage of the antibody construct is ≧80%, more preferably ≧85%, even more preferably ≧90% and most preferably ≧95%.

[0200] In one embodiment, the antibody construct preferably has a plasma stability (ratio of EC50 in the presence of plasma to EC50 in the absence of plasma) of ≦5 or ≦4, more preferably ≦3.5 or ≦3, even more preferably ≦2.5 or ≦2, and most preferably ≦1.5 or ≦1. The plasma stability of the antibody construct is determined by incubating the construct in human plasma at 37° C. for 24 hours, followed by 51The antibody constructs can be tested by determining the EC50 in a chromium release cytotoxicity assay. The effector cells in the cytotoxicity assay can be stimulated enriched human CD8 positive T cells. The target cells can be, for example, CHO cells transfected with human MUC17. The effector cell to target cell (E:T) ratio can be selected as 10:1 or 5:1. The human plasma pool used for this purpose is derived from blood of healthy donors collected by EDTA-coated syringes. The cellular components are removed by centrifugation and the upper plasma phase is collected and then pooled. As a control, the antibody constructs are diluted in RPMI-1640 medium immediately before the cytotoxicity assay. The plasma stability is calculated as the ratio of EC50 (after plasma incubation) to EC50 (control).

[0201] It is further preferred that the antibody construct of the present invention has a low monomer to dimer conversion rate. The conversion rate can be measured under different conditions and analyzed by high-performance size exclusion chromatography. For example, incubation of the monomeric isoform of the antibody construct can be performed for 7 days at 37°C in an incubator at a concentration of, for example, 100 μg / ml or 250 μg / ml. Under these conditions, it is preferred that the antibody construct of the present invention exhibits a dimer percentage of ≦5%, more preferably ≦4%, even more preferably ≦3%, even more preferably ≦2.5%, even more preferably ≦2%, even more preferably ≦1.5% and most preferably ≦1% or ≦0.5% or even 0%.

[0202] It is also preferred that the bispecific antibody construct of the present invention exhibits a very low dimer conversion rate after several freeze / thaw cycles. For example, the monomer of the antibody construct is adjusted to a concentration of, for example, 250 μg / ml in a universal formulation buffer and subjected to three freeze / thaw cycles (freezing at -80°C for 30 minutes followed by thawing at room temperature for 30 minutes) followed by rapid SEC to determine the percentage of the initial monomeric antibody construct that has been converted to a dimeric antibody construct. Preferably, the percentage of dimers in the bispecific antibody construct is, for example, ≦5%, more preferably ≦4%, even more preferably ≦3%, even more preferably ≦2.5%, even more preferably ≦2%, even more preferably ≦1.5% and most preferably ≦1% or even ≦0.5% after three freeze / thaw cycles.

[0203] The bispecific antibody constructs of the present invention preferably exhibit good thermal stability with an aggregation temperature of ≧45° C. or ≧50° C., more preferably ≧52° C. or ≧54° C., even more preferably ≧56° C. or ≧57° C. and most preferably ≧58° C. or ≧59° C. From this point of view, the thermal stability parameters can be determined as follows: An antibody solution with a concentration of 250 μg / ml is transferred into a single-use cuvette and placed in a dynamic light scattering (DLS) instrument. The sample is heated from 40° C. to 70° C. at a heating rate of 0.5° C. / min while the measurement radius is constantly acquired. The increase in radius, which indicates the melting and aggregation of the protein, is used to calculate the aggregation temperature of the antibody.

[0204] Alternatively, melting temperature curves can be determined by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of the antibody construct. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC instrument. The energy uptake of samples containing the antibody construct is recorded from 20°C to 90°C and compared to samples containing only the formulation buffer. The antibody construct is adjusted to a final concentration of, for example, 250 μg / ml in SEC running buffer. The overall temperature of the samples is increased stepwise to record each melting curve. The energy uptake of the samples and formulation buffer standards at each temperature T is recorded. The difference in energy uptake Cp (kcal / mole / °C) of the samples minus the standards is plotted against each temperature. The melting temperature is defined as the temperature at which the energy uptake first reaches a maximum.

[0205] It is also envisaged that the MUC17xCD3 bispecific antibody constructs of the invention have a turbidity (measured by OD340 after concentrating the purified monomeric antibody construct to 2.5mg / ml and incubating overnight) of ≦0.2, preferably ≦0.15, more preferably ≦0.12, even more preferably ≦0.1 and most preferably ≦0.08.

[0206] In a further embodiment, the antibody construct according to the present invention is stable at physiological pH or slightly lower pH, i.e., about pH 7.4-6.0. The higher the resistance of the antibody construct at non-physiological pH, e.g., about pH 6.0, the higher the recovery of the antibody construct eluted from the ion exchange column relative to the total amount of loaded protein. The recovery of the antibody construct from the ion (e.g., cation) exchange column at about pH 6.0 is preferably ≧30%, more preferably ≧40%, more preferably ≧50%, even more preferably ≧60%, even more preferably ≧70%, even more preferably ≧80%, even more preferably ≧90%, even more preferably ≧95% and most preferably ≧99%.

[0207] It is further envisaged that the bispecific antibody constructs of the invention exhibit therapeutic efficacy or anti-tumour activity, which can be assessed, for example, in the test disclosed in the generalised examples below in advanced stage human tumour xenograft models.

[0208] On the first day of the test, 5 x 10 6 Cells of a human target cell antigen (herein MUC17) positive cancer cell line are injected subcutaneously into the right dorsal flank of female NOD / SCID mice. The mean tumor volume is approximately 100 mm 3 When the total number of CD3+ T cells in the human body reaches 10, the in vitro expanded human CD3+ T cells are injected into the peritoneal cavity of the animals at approximately 2 × 10 7 Mice are implanted in mice by injection of 100 cells. Mice in vehicle control group 1 do not receive effector cells and are used as non-implanted controls for comparison with vehicle control group 2 (receiving effector cells) to monitor the effect of T cells alone on tumor growth. The mean tumor volume was approximately 200 mm 3 Antibody treatment begins when tumor volume reaches 100 μg / mL. The mean tumor size of each treatment group on the day treatment begins should not be statistically different from any other group (analysis of variance). Mice are treated with 0.5 mg / kg / day of the MUC17×CD3 bispecific antibody construct by intravenous bolus injection for approximately 15-20 days. Tumors are measured by caliper during the study and progression is assessed by between-group comparison of tumor volume (TV). Tumor growth inhibition T / C [%] is calculated as T / C% = 100 × (median TV of analyzed groups) / This is determined by calculating the TV as (median TV of control group 2).

[0209] The skilled artisan knows how to vary or adapt certain parameters of this test, such as the number of tumor cells injected, the injection site, the number of human T cells implanted, the amount of bispecific antibody construct administered and the timeline, and still obtain meaningful and reproducible results. Preferably, the tumor growth inhibition T / C[%] is ≦70 or ≦60, more preferably ≦50 or ≦40, even more preferably ≦30 or ≦20 and most preferably ≦10 or ≦5 or even ≦2.5. Preferably, the tumor growth inhibition is close to 100%.

[0210] In a preferred embodiment of the antibody construct of the present invention, the antibody construct is a single chain antibody construct. In a preferred embodiment of the antibody construct of the present invention, the third domain comprises, in order from amino to carboxyl: Hinge-CH2-CH3-Linker-Hinge-CH2-CH3 Includes.

[0211] In one embodiment of the invention, each of the polypeptide monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 24. In a preferred embodiment or invention, each of the polypeptide monomers has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 24.

[0212] Also, in one embodiment of the invention, the CH2 domain of one or preferably each (both) polypeptide monomer of the third domain comprises an intradomain cysteine ​​disulfide bridge. As known in the art, the term "cysteine ​​disulfide bridge" refers to a functional group with the general structure RSSR. This link, also called SS bond or disulfide bridge, is obtained by coupling of two thiol groups of cysteine ​​residues. With regard to the antibody construct of the invention, it is particularly preferred that the cysteines forming the cysteine ​​disulfide bridge in the mature antibody construct are introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering).

[0213] In one embodiment of the invention, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid other than Q. Said substitution is preferably N314G. In a more preferred embodiment, said CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0214] For example, it is believed that the preferred features of the antibody constructs of the invention compared to bispecific hetero-Fc antibody constructs known in the art (Figure 1b) may relate inter alia to the introduction of the above mentioned modifications in the CH2 domain. Thus, with regard to the constructs of the invention, it is preferred that the CH2 domain within the third domain of the antibody construct of the invention comprises intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321 and / or the glycosylation site at Kabat position 314 is removed, preferably by a N314G substitution.

[0215] In a further preferred embodiment of the present invention, the CH2 domain in the third domain of the antibody construct of the present invention comprises intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321, and the glycosylation site at Kabat position 314 is eliminated by a N314G substitution. The peptide monomer has an amino acid sequence selected from the group consisting of SEQ ID NOs:17 and 18.

[0216] In one embodiment, the present invention provides an antibody construct comprising: (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) An antibody construct is provided, wherein the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain.

[0217] Thus, the first and second domains may each be binding domains comprising two antibody variable domains, such as a VH and a VL domain. Examples of such binding domains comprising two antibody variable domains as described herein above include, for example, the Fv fragment, scFv fragment or Fab fragment as described herein above. Alternatively, either or both of the binding domains may comprise only a single variable domain. Examples of such single domain binding domains as described herein above include, for example, nanobodies or single variable domain antibodies comprising only one variable domain, which may be a VHH, VH or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0218] In a preferred embodiment of the antibody construct of the invention, the first and second domains are fused to the third domain via a peptide linker. Preferred peptide linkers are described herein above and are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 1) or a polymer thereof, i.e. (Gly4Ser)x, where x is an integer of 1 or higher (e.g. 2 or 3). A particularly preferred linker for the fusion of the first and second domains to the third domain is shown in SEQ ID NO: 1.

[0219] In a preferred embodiment, the antibody construct of the invention comprises, in amino to carboxyl order: (a) the first domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) the second domain; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12; (e) a first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (g) a second polypeptide monomer of the third domain. It is characterized by including.

[0220] The antibody constructs of the invention comprise a first domain that binds to MUC17, preferably to the extracellular domain (ECD) of MUC17. In the context of the present invention, the term "binds to the extracellular domain of MUC17" is understood to mean that the binding domain binds to MUC17 expressed on the surface of a target cell. Thus, the first domain according to the invention binds to MUC17 expressed by a cell or cell line in which MUC17 is naturally expressed and / or to a cell or cell line that has been transformed or (stably or transiently) transfected with MUC17. Preferably, the first binding domain binds to MUC17 when expressed by a human or animal host. In a preferred embodiment, the first binding domain also binds to MUC17 when MUC17 is used as a "target" or "ligand" molecule in an in vitro binding assay such as BIAcore or Scatchard. A "target cell" can be any prokaryotic or eukaryotic cell that expresses MUC17 on its surface; preferably, the target cell is a cell that is part of the human or animal body, such as a specific MUC17-expressing cancer or tumor cell.

[0221] Preferably, the first binding domain binds to human MUC17 / MUC17 ECD. In a more preferred embodiment, it binds to macaque MUC17 / MUC17 ECD. According to a most preferred embodiment, it binds to both human and macaque MUC17 / MUC17 The MUC17 extracellular domain binds to both the extracellular domain and the intracellular domain of MUC17. "MUC17 extracellular domain" or "MUC17 ECD" refers to a region or sequence of MUC17 that is essentially free of the transmembrane and intracytoplasmic domains of MUC17. It will be understood by those of skill in the art that the transmembrane domains identified for the MUC17 polypeptides of the invention are identified according to criteria routinely used in the art for identifying hydrophobic domains of that type. The exact boundaries of a transmembrane domain may vary, but are most likely no more than about 5 amino acids on either end of the domain specifically referred to herein.

[0222] Preferred binding domains that bind to MUC17 are disclosed in WO 2010 / 037836 and WO 2011 / 121110. Any binding domain for MUC17 described in these applications may be used in the context of the present invention.

[0223] In one aspect of the invention, the antibody construct comprises, in amino to carboxyl order: (a) a first domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 56, 68, 74, 86, 92, 104, 110, 122, 128, 140, 146, 158, 164, 176, 182, 194, 200, 212, 218, 230, 236, 248, 254, 266, 272, 284, 290, 302, 308, 320, 335, 350, 365, 380, 395, 410, 425, 440, 455, 470; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) a second domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 of WO 2008 / 119567, or as set forth in SEQ ID NO: 15; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12; (e) a first polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17-24; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (g) a second polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17 to 24; Includes.

[0224] In accordance with this preferred embodiment, the first and second domains fused to the single chain polypeptide via a peptide linker are selected from the group consisting of SEQ ID NOs: 51, 57, 69, 75, 87, 93, 105, 111, 123, 129, 141, 147, 159, 165, 177, 183, 195, 201, 213, 219, 231, 237, 249, 255, 267, 273, 285, 291, 303, 309, 321, 324, 336, 339, 351, 354, 366, 369, 381, 384, 396, 399, 411, 414, 426, 429, 441, 444, 456, 459, 471 and 474.

[0225] In one embodiment, the antibody construct of the invention comprises the antibody of SEQ ID NO: 52, 53, 58, 59, 70, 71, 76, 77, 88, 89, 94, 95, 106, 107, 112, 113, 124, 125, 130, 131, 142, 143, 148, 149, 160, 161, 166, 167, 178, 179, 184, 185, 196, 197, 202, 203, 214, 215, 220, 221, 232, 233, 238, 239, 250, 251, 256, 257, 268, 269, 274, 275, 286, 287, 292 , 293, 304, 305, 310, 311, 322, 323, 325, 326, 337, 338, 340, 341, 352, 353, 355, 356, 367, 368, 370, 371, 382, ​​383, 385, 386, 397, 398, 400, 401, 412, 413, 415, 416, 427, 428, 430, 431, 442, 443, 445, 446, 457, 458, 460, 461, 472, 473, 475 and 476.

[0226] The present invention further provides a polynucleotide / nucleic acid molecule encoding the antibody construct of the present invention. A polynucleotide is a biopolymer composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotides with different biological functions. Nucleotides are organic molecules that function as monomers or subunits of nucleic acid molecules such as DNA or RNA. A nucleic acid molecule or polynucleotide can be double-stranded and single-stranded, linear and circular. It is preferably contained within a vector contained within a host cell. The host cell is capable of expressing the antibody construct, for example after transformation or transfection with a vector or polynucleotide of the present invention. For that purpose, the polynucleotide or nucleic acid molecule is operably linked to a control sequence.

[0227] The genetic code is the set of rules that translate the information coded in genetic material (nucleic acids) into proteins. Biological decoding in living cells is performed by ribosomes, which carry amino acids and attach them in the order specified by the mRNA using tRNA molecules that read the mRNA three nucleotides at a time. This code defines how triplet nucleotide sequences called codons specify the amino acid that will be added next during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies one amino acid. Because most genes are coded with the exact same code, this particular code is often referred to as the canonical or standard genetic code. The genetic code determines the protein sequence of a given coding region, but other genomic regions can affect when and where these proteins are produced.

[0228] Furthermore, the present invention provides vectors comprising the polynucleotide / nucleic acid molecules of the present invention. A vector is a nucleic acid molecule used as a vehicle to transfer (foreign) genetic material into a cell. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. In general, genetically engineered vectors contain an origin of replication, a multiple cloning site, and a selection marker. The vector itself is generally a nucleotide sequence, generally a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. In addition to the transgene insert and backbone, modern vectors may include additional features such as promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags, etc. Vectors, called expression vectors (expression constructs), are specifically intended for the expression of a transgene in a target cell and generally have regulatory sequences.

[0229] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0230] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a protein precursor that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0231] "Transfection" is a method for the deliberate introduction of a nucleic acid molecule or polynucleotide (including a vector) into a target cell. The term is most often used for non-viral methods in eukaryotic cells. Transduction is often used to describe the viral-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells usually involves creating transient pores or "holes" in the cell membrane to allow uptake of a substance. Transfection can be performed using calcium phosphate, by electroporation, by cell compression, or by mixing cationic lipids with a substance to create liposomes that fuse with the cell membrane and accumulate the cargo inside.

[0232] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and into non-animal eukaryotic cells, including plant cells. Transformation is thus the genetic modification of bacteria or non-animal eukaryotic cells resulting from the direct uptake and subsequent incorporation of exogenous genetic material (nucleic acid molecules) from its surroundings through the cell membrane. Transformation can occur by artificial means. For transformation to occur, cells or bacteria must be in a competent state in which transformation can occur as a timed response to environmental conditions such as starvation and cell density.

[0233] Furthermore, the present invention provides host cells transformed or transfected with the polynucleotide / nucleic acid molecule or vector of the present invention. As used herein, the term "host cell" or "recipient cell" is intended to include any individual cell or cell culture that can be or has been a recipient of vectors encoding the antibody construct of the present invention, exogenous nucleic acid molecules and polynucleotides; and / or the antibody construct itself. Introduction of the respective substance into the cell is performed by transformation, transfection, etc. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Since certain modifications may occur in successive generations, either due to spontaneous, accidental or deliberate mutations, or due to environmental influences, such progeny may not in fact be completely identical (morphologically or in terms of genome or total DNA set) to the parent cell, but still fall within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, and include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque or human cells.

[0234] The antibody construct of the present invention can be produced in bacteria. After expression, the antibody construct of the present invention can be isolated from the E. coli cell paste in a soluble fraction and purified, for example, by affinity chromatography and / or size exclusion. Final purification can be performed, for example, similar to the purification method for antibodies expressed in CHO cells.

[0235] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the antibody constructs of the present invention. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly available and useful in the present invention, such as Schizosaccharomyces pombe, K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 16045), K. saccharomyces pombe ... Kluyveromyces hosts such as Kluyveromyces spp., K. thermotolerans and K. marxianus; Yarrowia spp. (EP 402226); Pichia pastoris (EP 183070); Candida spp.; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces occidentalis; occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

[0236] Suitable host cells for the expression of the glycosylated antibody constructs of the present invention are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0237] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors useful for the production of proteins in plant cell culture are known to those of skill in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Owen et al., Nature (1989) 342:76-78; et al. (1992) Bio / Technology 10:790-794, Artsaenko et al. (1995) The Plant J 8:745-750 and Fecker et al. (1996) Plant Mol Biol 32 Please see: 979-986.

[0238] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0239] In a further embodiment, the present invention provides a method for the production of an antibody construct of the present invention, the method comprising culturing a host cell of the present invention under conditions allowing expression of the antibody construct of the present invention and recovering the produced antibody construct from the culture.

[0240] As used herein, the term "culturing" refers to the maintenance, differentiation, growth, proliferation and / or propagation of cells in vitro under suitable conditions in a culture medium. The term "expression" includes any step involved in the production of an antibody construct of the invention, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0241] When using recombinant techniques, the antibody constructs can be produced intracellularly in the periplasmic space or directly secreted into the medium. If the antibody constructs are produced intracellularly, the first step is to remove particulate debris of host cells or lysed fragments, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminating bacteria.

[0242] The antibody constructs of the invention prepared from host cells can be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. Depending on the antibody to be recovered, other protein purification techniques may be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, anionic or Alternatively, chromatography on a cation exchange resin (e.g., a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation can be used. When the antibody construct of the present invention contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) is useful for purification.

[0243] Affinity chromatography is the preferred purification technique. The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.

[0244] Furthermore, the present invention provides a pharmaceutical composition comprising the antibody construct of the present invention or the antibody construct produced according to the method of the present invention. In the pharmaceutical composition of the present invention, the homogeneity of the antibody construct is preferably ≧80%, more preferably ≧81%, ≧82%, ≧83%, ≧84% or ≧85%, even more preferably ≧86%, ≧87%, ≧88%, ≧89% or ≧90%, even more preferably ≧91%, ≧92%, ≧93%, ≧94% or ≧95%, and most preferably ≧96%, ≧97%, ≧98% or ≧99%.

[0245] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention comprise one or more antibody constructs of the present invention, preferably in a therapeutically effective amount. Preferably, the pharmaceutical composition further comprises a suitable formulation of one or more (pharmaceutical effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. Components of acceptable compositions are preferably non-toxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0246] The composition of the present invention may include a pharmaceutically acceptable carrier.Generally, as used herein, "pharmaceutically acceptable carrier" refers to any aqueous and non-aqueous solution, sterile solution, solvent, buffer solution, such as phosphate buffered saline (PBS) solution, water, suspension, emulsion such as oil / water emulsion, various types of wetting agents, liposome, dispersion medium and coating that are suitable for pharmaceutical administration, especially parenteral administration.The use of such media and agents in pharmaceutical compositions is well known in the art, and the composition containing such carriers can be formulated by well known conventional methods.

[0247] Certain embodiments provide pharmaceutical compositions comprising an antibody construct of the invention and further one or more excipients, such as those exemplarily described in this section and elsewhere herein. Excipients can be used in the invention for a wide range of purposes, including methods of the invention for adjusting the physical, chemical or biological properties of the formulations, such as adjusting the viscosity, and / or improving the effectiveness and / or stabilizing such formulations, as well as against degradation and damage due to stresses encountered during and after manufacture, transportation, storage, preparation prior to use, administration.

[0248] In certain embodiments, a pharmaceutical composition may contain formulation materials intended to, for example, modify, sustain, or protect the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition (REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company). ny). In such embodiments, suitable formulation materials may include, but are not limited to: Charged amino acids, preferably containing lysine, lysine acetate, arginine, glutamate and / or histidine, e.g. glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine Antibacterial and antifungal agents ·Antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium bisulfite; buffers, buffer systems and buffering agents used to maintain the composition at physiological pH or slightly lower, preferably at a lower pH of 4.0 to 6.5; examples of buffers are borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids, succinate, phosphate and histidine; for example, Tris buffer at about pH 7.0 to 8.5; non-aqueous solvents, for example propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; Aqueous carriers, including water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media; · Biodegradable polymers such as polyester; ·Bulking agents such as mannitol or glycine; · Chelating agents such as ethylenediaminetetraacetic acid (EDTA); ·Isotonicity and absorption retarding agents; complexing agents, e.g. caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin) Injectables; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol; (low molecular weight) proteins, polypeptides or proteinaceous carriers, such as human or bovine serum albumin, gelatin or immunoglobulins, preferably of human origin; Colouring and flavouring agents; Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol and sodium thiosulfate · Diluents; ·emulsifier; -Hydrophilic polymers such as polyvinylpyrrolidone) · Salt-forming counterions such as sodium; preservatives, such as antimicrobials, antioxidants, chelating agents, and inert gases; examples are benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); Metal complexes such as Zn-protein complexes; Solvents and co-solvents (such as glycerin, propylene glycol or polyethylene glycol); sugars and sugar alcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbose, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclitols (e.g. inositol), polyethylene glycol; and polyhydric sugar alcohols; ·Suspending agents; Surfactants or wetting agents, such as Pluronic®, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate, Triton, trometa amine, lecithin, cholesterol, tyloxapal; the surfactant may be a detergent, preferably with a molecular weight of >1.2KD and / or a polyether, preferably with a molecular weight of >3KD; non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000; ·Stability enhancers such as sucrose or sorbitol; isotonicity enhancing agents, such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol; Parenteral delivery vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils; Intravenous delivery vehicles, including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0249] In the context of the present invention, a pharmaceutical composition, which may be preferably a liquid composition, or may be a solid composition obtained by lyophilization, or may be a reconstituted liquid composition, comprises: (a) an antibody construct comprising at least three domains, the first domain binds to a surface antigen of a target cell and has an isoelectric point (pI) in the range of 4 to 9.5; the second domain binds to a second antigen and has a pI in the range of 8 to 10, preferably 8.5 to 9.0; and optionally, the third domain comprises two polypeptide monomers, each of which comprises a hinge, a CH2 domain and a CH3 domain, the two polypeptide monomers being fused to each other via a peptide linker; (b) at least one buffering agent; (c) at least one saccharide; and (d) at least one surfactant and the pH of the pharmaceutical composition is in the range of 3.5 to 6.

[0250] It is further envisaged in the context of the present invention that the at least one buffering agent is present in a concentration range of 5 to 200 mM, more preferably in a concentration range of 10 to 50 mM. It is envisaged in the context of the present invention that the at least one sugar is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextrans or linear unbranched dextrans. It is also envisaged in the context of the present invention that the disaccharide is selected from the group consisting of sucrose, trehalose and mannitol, sorbitol and combinations thereof. It is further envisaged in the context of the present invention that the sugar alcohol is sorbitol. It is envisaged in the context of the present invention that the at least one sugar is present in a concentration range of 1 to 15% (m / V), preferably in a concentration range of 9 to 12% (m / V).

[0251] It is also envisaged in the context of the present invention that the at least one surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, Pluronic® F68, Triton X-100, polyoxyethylene, PEG 3350, PEG 4000 and combinations thereof. It is further envisaged in the context of the present invention that the at least one surfactant is present in a concentration in the range of 0.004-0.5% (m / V), preferably in the range of 0.001-0.01% (m / V). It is further envisaged in the context of the present invention that the pH of the composition is in the range of 4.0-5.0, preferably 4.2. It is also envisaged in the context of the present invention that the pharmaceutical composition has an osmolality in the range of 150-500 mOsm. It is further envisaged in the context of the present invention that the pharmaceutical composition further comprises an excipient selected from the group consisting of one or more polyols and one or more amino acids. It is envisaged in the context of the present invention that said one or more excipients are present in a concentration range of 0.1-15% (w / V).

[0252] The pharmaceutical composition comprises (a) an antibody construct as described above, (b) 10 mM glutamate or acetate; (c) 9% (m / V) sucrose or 6% (m / V) sucrose and 6% (m / V) hydroxypropyl-β-cyclodextrin; (d) 0.01% (m / V) polysorbate 80 and wherein the pH of the liquid pharmaceutical composition is 4.2.

[0253] It is further envisaged in the context of the present invention that the antibody construct is present in a concentration range of 0.1-8 mg / ml, preferably 0.2-2.5 mg / ml, more preferably 0.25-1.0 mg / ml.

[0254] It will be apparent to one skilled in the art that different components of a pharmaceutical composition (e.g., those listed above) may have different effects, e.g., amino acids can act as buffers, stabilizers and / or antioxidants; mannitol can act as a bulking agent and / or tonicity enhancer; sodium chloride can act as a delivery vehicle and / or tonicity enhancer, etc.

[0255] It is envisaged that the compositions of the invention may contain, in addition to the polypeptides of the invention as defined herein, further biologically active agents depending on the intended use of the composition. Such agents may be drugs acting on the gastrointestinal system, drugs acting as cytostatic agents, drugs preventing hyperurikemia, drugs inhibiting immune responses (e.g. corticosteroids), drugs modulating inflammatory responses, drugs acting on the circulatory system and / or cytokines, etc., known in the art. It is also envisaged that the antibody constructs of the invention are applied in combination therapy, i.e. in combination with another anti-cancer drug.

[0256] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vitro clearance rate of the antibody construct of the present invention. In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the antibody construct of the composition of the present invention may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing the selected composition having the desired degree of purity, optionally with a compounding agent (REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Further, in certain embodiments, the antibody constructs of the present invention may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0257] When parenteral administration is contemplated, the therapeutic composition for use in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired antibody construct of the present invention in a pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antibody construct of the present invention is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the formulation can be delivered via depot injection. These may include formulations of the desired molecule with agents that can provide controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid may also be used, which has the effect of enhancing sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired antibody construct.

[0258] Further pharmaceutical compositions will be evident to those skilled in the art, including formulations comprising the antibody constructs of the present invention in sustained or controlled delivery / release formulations. Techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US 93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained release formulations may include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. The sustained release matrix may comprise polyesters, hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919, EP 058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (EP 133,988). The sustained release composition may also comprise liposomes, which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Applications 036,676; 088,046 and 143,949.

[0259] The antibody constructs may also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. Ed. (1980).

[0260] Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are generally filled into containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be pierced by hypodermic injection needles.

[0261] Another aspect of the invention includes the self-buffering antibody constructs of the formulations of the invention that can be used as pharmaceutical compositions, as described in International Patent Application WO 06138181 A2 (PCT / US 2006 / 022599). Various descriptions of protein stabilization and formulation materials and methods useful in this regard are available, see, for example, Arakawa et al., "Solvent interactions in pharmaceutical formulations", Pharm Res. 8(3):285-91 (1991); Kendrick et al. .,"Physical stabilization of proteins in aqueous solution” in:RATIONAL DESIGN OF See STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002) and Randolph et al., "Surfactant-protein interactions", Pharm Biotechnol. 13:159-75 (2002), particularly with respect to protein pharmaceuticals and processes for veterinary and / or human medical use, and in particular the portions relating to the same excipients and processes as the self-buffering protein formulations according to the present invention.

[0262] Salts may be used according to certain embodiments of the invention, for example, to adjust the ionic strength and / or tonicity of the formulation and / or to improve the solubility and / or physical stability of proteins or other components of the compositions according to the invention. As is well known, ions can stabilize proteins in the native state by binding to charged residues on the surface of the protein and by shielding charged and polar groups in the protein, reducing the strength of their electrostatic, attractive and repulsive interactions. Ions can also stabilize proteins in the denatured state, in particular by binding to modified peptide bonds (--CONH) of proteins. Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0263] Different ionic species have significantly different effects on proteins. Several taxonomic rankings of ions and their effects on proteins have been proposed and can be used in formulating pharmaceutical compositions according to the invention. One example is the Hofmeister series, which ranks ionic and polar non-ionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropic". Destabilizing solutes are called "chaotropic". Kosmotropes are commonly used in high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins out of solution ("salting out"). Chaotropes are commonly used to denature and / or solubilize proteins ("salting in"). The relative effects of an ion on "salting in" and "salting out" define the ion's position in the Hofmeister series.

[0264] Free amino acids can be used in the antibody constructs of the formulations of the invention according to various embodiments of the invention as bulking agents, stabilizers and antioxidants, as well as other standard uses. Lysine, proline, serine and alanine can be used to stabilize proteins in the formulation. Glycine is useful for ensuring proper cake structure and properties upon lyophilization. Arginine can be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0265] Polyols include sugars such as mannitol, sucrose, and sorbitol, and polyhydric alcohols such as glycerol and propylene glycol, and for purposes of discussion herein, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. They are useful stabilizers to protect proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful to adjust the isotonicity of the formulation. Among the polyols useful in selected embodiments of the invention is mannitol, which is commonly used in lyophilized formulations to ensure structural stability of the cake. Mannitol ensures structural stability of the cake. Generally, it is used in conjunction with a lyoprotectant, such as sucrose. Sorbitol and sucrose are used as agents to adjust isotonicity and to protect the protein from freezing during shipping or bulk preparation in the manufacturing process. Polyols that are preferred for use in the present invention include stabilizing agents for protection against freeze-thaw stress. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they are generally not included among the preferred polyols for use in the present invention. In addition, sugars that form such reactive species, such as sucrose, are also not included among the preferred polyols of the present invention in that they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a cryoprotectant, and can be used in the present invention in this regard.

[0266] The antibody construct embodiment of the formulation of the present invention further comprises a surfactant. Protein molecules can be prone to surface adsorption and denaturation and resulting aggregation at air-liquid, solid-liquid and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These adverse interactions are generally inversely proportional to protein concentration and are usually exacerbated by physical agitation, such as that encountered during product transportation and handling. Surfactants are routinely used to prevent, minimize or reduce surface adsorption. Surfactants useful in the present invention in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate and poloxamer 188. Surfactants are also commonly used to control protein conformational stability. In this regard, the use of surfactants is protein specific, since any given surfactant will usually stabilize some proteins and destabilize others.

[0267] Polysorbates are prone to oxidative degradation and often contain sufficient peroxide content as supplied to cause oxidation of the side chains of protein residues, especially methionine. Polysorbates should therefore be used with caution and, when used, at the lowest possible concentrations. In this respect, polysorbates exemplify the general rule that excipients should be used at the lowest possible concentrations.

[0268] The antibody construct embodiment of the formulation of the present invention further comprises one or more antioxidants. By maintaining an appropriate level of ambient oxygen and temperature, and by avoiding exposure to light, harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent. Antioxidant excipients can be used to prevent oxidative degradation of proteins as well. Antioxidants that are particularly useful in this regard include reducing agents, oxygen / free radical scavengers and chelating agents. Antioxidants for use in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain activity over the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention. Antioxidants can damage proteins. For example, reducing agents such as glutathione can break intramolecular disulfide bonds, among others. Thus, antioxidants for use in the present invention are selected, inter alia, to eliminate or sufficiently reduce the possibility of damaging proteins in the formulation themselves.

[0269] The formulations according to the invention may contain metal ions, which are cofactors for proteins and are required to form protein coordination compounds, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some processes that break down proteins. However, metal ions also catalyze physical and chemical processes that break down proteins. Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +2 , Mn +2 and Zn +2 can destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, which is +2 , Mn +2 and Zn + 2 , Cu +2 and Fe +2The aggregation can be destabilized by Al +3 It can be increased by ions.

[0270] The antibody construct embodiment of the formulation of the present invention further comprises one or more preservatives. Preservatives are necessary when developing a multi-dose parenteral formulation with more than one withdrawal from the same container. Its main function is to inhibit microbial growth and ensure sterility of the product over the shelf life or use period of the formulation. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use with small molecule parenteral drugs, developing protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is the main factor limiting their use in multi-dose protein formulations. To date, most protein drugs have only been formulated for single use. However, if a multi-dose formulation were possible, it would add the benefits of patient convenience and increased marketability. A good example is human growth hormone (hGH), where the development of a preserved formulation led to the development of a more convenient multi-use injection pen for commercialization. At least four such pen devices containing preserved formulations of hGH are currently available on the market: Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech) and Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol. During the formulation and development of preserved dosage forms, several aspects need to be considered. The effective preservative concentration in the formulation must be optimized. This requires testing a given preservative in the dosage form over a range of concentrations that impart antimicrobial effect without compromising protein stability.

[0271] As expected, developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with a preservative containing diluent at the time of use. This reduces the time the preservative is in contact with the protein and greatly minimizes the associated stability risks. For liquid formulations, the efficacy and stability of the preservative should be maintained throughout the entire product shelf life (approximately 18-24 months). It is important to note that the efficacy of the preservative needs to be demonstrated in the final formulation containing the active drug and all excipient components.

[0272] The antibody constructs disclosed herein may also be formulated as immunoliposomes. "Liposomes" are small vesicles composed of various types of lipids, phospholipids and / or surfactants that are useful for drug delivery to mammals. The components of the liposome are generally arranged in a bilayer structure similar to the lipid arrangement of biological membranes. Liposomes containing antibody constructs are prepared by methods known in the art, for example, as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 97 / 38731. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody constructs of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al. J. Biol. Chem. 257:286-288 (1982). Optionally, a chemotherapeutic agent can be included within the liposomes. See Gabizon et al. J. National Cancer Inst. 81(19)1484(1989).

[0273] After the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized).

[0274] The biological activity of the pharmaceutical compositions defined herein may be determined, for example, by cytotoxicity assays as described in the Examples below, in WO 99 / 54440, or in Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12). As used herein, "efficacy" or "in vivo efficacy" refers to the response to therapy with the pharmaceutical composition of the present invention, for example, using standardized NCI response criteria. The success of therapy with the pharmaceutical composition of the present invention or in vivo efficacy refers to the efficacy of the composition for its intended purpose, i.e., the ability of the composition to cause its desired effect, i.e., depletion of pathological cells, e.g., tumor cells. In vivo efficacy may be monitored by established standard methods for the respective disease entity, including, but not limited to, white blood cell counts, differential, fluorescence activated cell sorting, bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used.In addition, computed tomography, x-ray, and nuclear magnetic resonance imaging (e.g., response assessment based on the National Cancer Institute criteria [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkins lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999 Apr;17(4):1244]), positron emission tomography scanning, white blood cell count, differential, fluorescence activated cell sorting, bone marrow aspiration, lymph node biopsy / histology and various lymphoma-specific clinical chemistry parameters (e.g., lactate dehydrogenase) and other established standard methods may be used.

[0275] Another major challenge in the development of drugs such as the pharmaceutical compositions of the present invention is the predictable modulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of a candidate drug can be established, i.e., a profile of pharmacokinetic parameters that affect the ability of a particular drug to treat a given pathology. Pharmacokinetic parameters of a drug that affect the ability of a drug to treat a particular disease entity include, but are not limited to, half-life, volume of distribution, hepatic first-pass metabolism, and degree of serum binding. The efficacy of a given drug can be influenced by each of the above parameters. A presumed feature of the antibody constructs of the present invention provided by a particular FC format is that they include differences, for example, in pharmacokinetic behavior. The half-life extended targeting antibody constructs of the present invention preferably exhibit a surprisingly increased residence time in vivo compared to the "canonical" non-HLE version of said antibody construct.

[0276] "Half-life" refers to the time it takes for 50% of an administered drug to be eliminated through biological processes, such as metabolism, excretion, etc. "Hepatic first-pass metabolism" refers to the tendency of a drug to be metabolized upon first contact with the liver, i.e., during its first passage through the liver. "Volume of distribution" refers to, e.g. "Extent of serum binding" refers to the degree of retention of a drug across various compartments of the body, such as intracellular and extracellular spaces, tissues and organs, and the distribution of the drug within these compartments. "Extent of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins, such as albumin, resulting in a reduction or elimination of the drug's biological activity.

[0277] Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rate, onset of more action and / or Cmax for a given amount of drug administered. "Bioavailability" refers to the amount of drug in the blood compartment. "Lag time" refers to the delay time from administration of a drug until it can be detected and measured in blood or plasma. "Tmax" is the time after which the maximum blood concentration of the drug is reached, and "Cmax" is the maximum blood concentration achieved by a given drug. All parameters affect the time it takes for a drug to reach the blood or tissue concentration required for a biological effect. Pharmacokinetic parameters of bispecific antibody constructs exhibiting cross-species specificity that can be determined in preclinical animal studies in non-chimpanzee primates as outlined above are also provided, for example, in the publication by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12).

[0278] In a preferred embodiment of the invention, the pharmaceutical composition is stable for at least 4 weeks at about -20°C. As is evident from the accompanying examples, the quality of the antibody constructs of the invention relative to the quality of the corresponding state-of-the-art antibody constructs can be tested using different systems. These tests are understood to be in accordance with the "ICH Harmonised Tripartite Guideline: Stability Testing of Biotechnological / Biological Products Q5C and Specifications: Test procedures and Acceptance Criteria for Biotech Biotechnological / Biological Products Q6B" and are selected to provide a stability indicator profile that allows reliable detection of changes in the identity, purity and potency of the product. It is well accepted that the term purity is a relative term. Due to the effects of glycosylation, deamidation or other heterogeneity, the absolute purity of a biotechnological / biological product should usually be assessed by more than one method and the purity values ​​obtained are method-dependent. For the purpose of stability testing, the tests of purity should be aligned with the method of determination of degradation products.

[0279] To assess the quality of a pharmaceutical composition comprising an antibody construct of the present invention, it can be analyzed, for example, by analyzing the content of soluble aggregates in solution (HMWS by size exclusion). Stability at about -20°C for at least 4 weeks is characterized by a content of less than 1.5% HMWS, preferably less than 1% HMWS.

[0280] A preferred formulation for the antibody construct as a pharmaceutical composition may, for example, include the components of the formulation as described below. ·formulation: Potassium phosphate pH 6.0, L-arginine hydrochloride, trehalose dihydrate, polysorbate 80 Further examples of the assessment of the stability of antibody constructs of the invention in the form of pharmaceutical compositions are provided in the accompanying Examples 4-12. In those Examples, embodiments of the antibody constructs of the invention are tested in different pharmaceutical formulations with respect to different stress conditions and the results are compared with other half-life extended (HLE) formats of bispecific T cell engaging antibody constructs known in the art. In general, antibody constructs provided in a particular FC format according to the invention will generally be compared with antibody constructs provided with different HLE formats and antibody constructs without any HLE format (e.g. "canonical" antibody constructs). It is assumed that the antibody construct is more stable against a wide range of stress conditions, such as temperature and light stress, compared to both the antibody construct and the cellular structure (the cellular structure). The aforementioned temperature stability may relate to both low temperatures (below room temperature, including freezing temperatures) and high temperatures (above room temperature, including temperatures up to or above body temperature). As those skilled in the art will appreciate, such improved stability against stresses that are difficult to avoid in clinical practice makes the antibody construct safer, since there will be fewer degradation products in clinical practice. As a result, the aforementioned improved stability means improved safety.

[0281] One embodiment provides an antibody construct of the invention, or an antibody construct made according to the method of the invention, for use in the prevention, treatment or amelioration of cancer associated with MUC17 expression or overexpression of MUC17, such as prostate cancer.

[0282] The formulations described herein are useful as pharmaceutical compositions for treating, ameliorating and / or preventing the pathological medical conditions described herein in patients in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes application or administration of the formulation to the body, isolated tissues or cells of a patient having a disease / disorder, a symptom of a disease / disorder or a predisposition to a disease / disorder with the intent to cure, remedy, relieve, alleviate, alter, correct, ameliorate, improve or affect the disease, symptom of a disease or predisposition to a disease.

[0283] As used herein, the term "amelioration" refers to any improvement in the disease state of a patient having a disease as set forth herein below, by administration of an antibody construct according to the present invention to a subject in need thereof. Such improvement may also be seen as a slowing or halting of progression of the patient's disease. As used herein, the term "prevention" refers to the avoidance of the onset or recurrence of a tumor or cancer or metastatic cancer as set forth herein below, by administration of an antibody construct according to the present invention to a subject in need thereof.

[0284] The term "disease" refers to any condition that would benefit from treatment with an antibody construct or pharmaceutical composition described herein, including chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in question.

[0285] A "neoplasm" is an abnormal growth of tissue, usually, but not necessarily, forming a mass. When it forms a mass, it is also commonly called a "tumor". A neoplasm or tumor can be benign, potentially malignant (precancerous) or malignant. Malignant neoplasms are generally called cancers. They usually invade and destroy surrounding tissues and can form metastases, i.e., they spread to other parts, tissues or organs of the body. Thus, the term "metastatic cancer" encompasses metastases to other tissues or organs other than that of the primary tumor. Lymphomas and leukemias are lymphatic neoplasms. For the purposes of the present invention, they are also encompassed by the terms "tumor" or "cancer".

[0286] The term "viral disease" refers to a disease that is the result of infection of a subject with a virus. As used herein, the term "immune disorder" refers to immune disorders such as autoimmune diseases, hypersensitivity disorders, and immune deficiencies in accordance with the general definition of the term.

[0287] In one embodiment, the invention provides a method for the treatment or amelioration of cancer associated with MUC17 expression or overexpression of MUC17, comprising administering to a subject in need thereof an antibody construct of the invention or an antibody construct made according to the method of the invention. The MUC17xCD3 bispecific single chain antibody is particularly advantageous for the treatment of cancer, preferably solid tumors, more preferably carcinoma and prostate cancer.

[0288] The term "subject in need" or "subject in need of treatment" refers to a subject who already has the disorder. Subjects in need or "patients" include humans and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0289] The antibody constructs of the present invention will generally be designed for a particular route and method of administration, a particular dosage and frequency of administration, and a particular treatment of a particular disease, particularly in the areas of bioavailability and duration. The materials of the composition are preferably formulated in concentrations that are acceptable to the site of administration.

[0290] Thus, formulations and compositions may be designed for delivery by any suitable route of administration in accordance with the present invention. Topical route (e.g. on the skin, inhalation, nose, eyes, auricle / ear, vagina, mucous membranes); Enteral routes (e.g., oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and Parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intra-articular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal) Including, but not limited to:

[0291] The pharmaceutical compositions and antibody constructs of the invention are particularly useful for parenteral administration, e.g., subcutaneous or intravenous delivery, e.g., by injection, such as a bolus injection, or by infusion, such as continuous infusion. The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0292] In particular, the present invention provides for uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e. continuous administration, may be achieved by a miniature pump system worn by the patient to regulate the inflow of therapeutic agent into the patient's body. The pharmaceutical composition comprising the antibody construct of the present invention may be administered by using said pump system. Such pump systems are generally known in the art and typically rely on periodic replacement of a cartridge containing the therapeutic agent to be infused. When replacing the cartridge in such a pump system, a momentary interruption in the otherwise uninterrupted inflow of therapeutic agent into the patient's body may result. Even in such a case, the administration step before the cartridge replacement and the administration step after the cartridge replacement would still be considered within the meaning of the pharmaceutical means and methods of the present invention, which together constitute the "uninterrupted administration" of such therapeutic agent.

[0293] The continuous or uninterrupted administration of the antibody construct of the present invention may be intravenous or subcutaneous administration by a fluid delivery device or miniature pump system, which includes a fluid delivery mechanism for pumping fluid from a reservoir and a drive mechanism for driving the delivery mechanism. The pump system for subcutaneous administration may include a needle or cannula for penetrating the patient's skin and delivering the suitable composition into the patient's body. The pump system may be fixed or attached directly to the patient's skin, whether vein, artery or blood vessel, allowing direct contact between the pump system and the patient's skin. The pump system may be attached to the patient's skin for 24 hours to several days. In some cases, the pump system may be a miniature pump system with a small reservoir volume. As a non-limiting example, the reservoir volume for the suitable pharmaceutical composition to be administered may be 0.1 to 50 ml.

[0294] Continuous administration can also be transdermal, by a patch that is attached to the skin and replaced from time to time.Those skilled in the art are aware of suitable patch systems for drug delivery for this purpose.It is noted that transdermal administration is particularly suitable for uninterrupted administration, since it has the advantage that, for example, a new second patch can be attached to the skin surface directly next to the first used patch, and the replacement of the first used patch can be completed at the same time just before removing the first used patch.There is no problem of interruption of inflow or battery failure.

[0295] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with a suitable liquid prior to administration. The lyophilized material may be reconstituted, for example, with bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was present prior to lyophilization.

[0296] The composition of the present invention can be administered to a subject at a suitable dose, which can be determined, for example, by a dose escalation study, by administering increasing doses of the antibody construct of the present invention, which exhibits cross-species specificity as described herein, to a non-chimpanzee primate, such as a macaque. As mentioned above, the antibody construct of the present invention, which exhibits cross-species specificity as described herein, has the advantage that it can be used in the same form in preclinical trials in non-chimpanzee primates and as a drug in humans. The administration schedule will be determined by the attending physician based on clinical factors. As is well known in the medical field, the dosage for a given patient depends on many factors, including the patient's size, body surface area, age, the individual compound administered, sex, time and route of administration, general health, and other drugs administered at the same time.

[0297] The term "effective dose" or "effective administration amount" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The amount or dose effective for this use will depend on the condition (indication) being treated, the antibody construct being delivered, the nature and purpose of the treatment, the severity of the disease, previous treatments, the patient's medical history and responsiveness to the therapeutic agent, the route of administration, the size (weight, body surface area or organ size) and / or condition (age and general health) of the patient, and the general condition of the patient's own immune system. The appropriate dose can be administered to the patient in a single dose or multiple doses, and can be adjusted according to the judgment of the attending physician to obtain the optimal therapeutic effect.

[0298] Typical dosages can range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors mentioned above. In certain embodiments, dosages can range from 1.0 μg / kg up to about 20 mg / kg, optionally 10 μg / kg up to about 10 mg / kg, or 100 μg / kg up to about 5 mg / kg.

[0299] A therapeutically effective amount of the antibody construct of the invention preferably reduces the severity of disease symptoms, increases the frequency or duration of disease symptom-free periods, or prevents impaired function or disability due to disease affliction. With respect to treating diseases associated with MUC17 expression as described herein above, a therapeutically effective amount of the antibody construct of the invention, the anti-MUC17 / anti-CD3 antibody construct herein, preferably inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated patients. The ability of a compound to inhibit tumor growth can be assessed in animal models predictive of efficacy.

[0300] The pharmaceutical composition can be administered in a single treatment or, if necessary, in combination with additional treatments, such as anti-cancer therapies, including other proteinaceous and non-proteinaceous drugs, which are administered simultaneously with the composition comprising the antibody construct of the invention as defined herein. The antibody construct may be administered either intracellularly or intracellularly, or may be administered separately at a predetermined time interval and dosage before or after administration of the antibody construct.

[0301] As used herein, the term "effective and non-toxic dose" refers to a tolerated dose of the antibody construct of the present invention that is sufficient to result in depletion of pathological cells, elimination of tumors, regression of tumors or stabilization of disease without or essentially without significant toxic effects. Such an effective and non-toxic dose can be determined, for example, by dose escalation studies as described in the art, and should be below the dose that induces serious adverse side events (dose-limiting toxicity, DLT).

[0302] As used herein, the term "toxicity" refers to the toxic effects of a drug that manifest as adverse events or serious adverse events. These side events may refer to a lack of systemic drug tolerance and / or a lack of local tolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0303] As used herein, the terms "safety", "in vivo safety" or "tolerability" are defined as administration of a drug that does not induce serious adverse events immediately after administration (local tolerance) and during longer drug application periods. "Safety", "in vivo safety" or "tolerability" can be evaluated, for example, periodically during treatment and follow-up periods. Measurements include clinical evaluations, such as organ findings and screening for laboratory abnormalities. Clinical evaluations can be performed and deviations from normal findings recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings can be evaluated, for example, according to the Common Terminology Criteria The criteria for adverse events v3.0 (CTCAE) may include allergy / immunology, blood / bone marrow, cardiac arrhythmia, coagulation, etc. Laboratory parameters that may be tested include, for example, hematology, clinical chemistry, coagulation profile and urinalysis as well as tests of other body fluids, such as serum, plasma, lymphatic or spinal fluid. Thus, safety may be assessed by, for example, physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scan, magnetic resonance imaging (MRI)), other measurements using technical devices (i.e. electrocardiogram), vital signs, measuring laboratory parameters and recording adverse events. For example, in the uses and methods according to the invention, adverse events in non-chimpanzee primates may be tested by histopathological and / or histochemical methods.

[0304] The above terms are also referenced, for example, in Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting of July 16, 1997.

[0305] Finally, the present invention provides kits comprising the antibody construct of the invention, the antibody construct produced according to the method of the invention, the pharmaceutical composition of the invention, the polynucleotide of the invention, the vector of the invention and / or the host cell of the invention.

[0306] In the context of the present invention, the term "kit" refers to two or more components packaged together in a container, vessel or other package, one of the components corresponding to an antibody construct, pharmaceutical composition, vector or host cell of the present invention. A kit can thus be described as a set of products and / or implements sufficient to accomplish a particular purpose that can be sold as a single item.

[0307] The kit may be of any suitable shape, size and material (preferably waterproof, e.g., waterproof) containing the antibody construct or pharmaceutical composition of the invention in a dosage amount suitable for administration (see above). The kit may comprise one or more containers (e.g., vials, ampoules, containers, syringes, bottles, bags) of any suitable material, e.g., plastic or glass. The kit may further comprise instructions for use (e.g., in the form of a leaflet or instruction manual), a means for administering the antibody construct of the invention, e.g., a syringe, pump, infuser, etc., a means for reconstituting the antibody construct of the invention, and / or a means for diluting the antibody construct of the invention.

[0308] The present invention also provides kits for single-dose administration units. The kits of the present invention may also include a first container containing a dried / lyophilized antibody construct and a second container containing an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0309] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "reagents" includes one or more of such different reagents, and reference to "methods" includes reference to equivalent steps and methods known to those of skill in the art that may be modified for or substituted for the methods described herein.

[0310] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0311] The term "and / or" wherever used in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0312] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range, however, the term also includes specific numbers, for example, about 20 includes 20.

[0313] The terms "less than" or "greater than" include a specific number. For example, less than 20 means less than or equal to. Similarly, greater than or greater than means greater than or equal to, or greater than or equal to, respectively.

[0314] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprise" can also be replaced with the terms "containing" or "including" or, when used herein, the term "having."

[0315] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0316] As used herein, in each instance, any of the terms "comprising," "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0317] All publications and patents cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference is inconsistent or inconsistent with the present specification, the present specification will take precedence over any such material.

[0318] A better understanding of the present invention and its advantages will be obtained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] 1. A bispecific antibody construct comprising: a first domain that binds to MUC17, and A second domain that binds to an extracellular epitope of the human and macaque CD3ε chain 1. A bispecific antibody construct comprising: [Aspect 2] 2. The bispecific antibody construct according to aspect 1, further comprising a third domain comprising two polypeptide monomers each comprising a hinge, a CH2 domain and a CH3 domain, said two polypeptide monomers being fused to each other via a peptide linker. [Aspect 3] 3. The bispecific antibody construct according to embodiment 1 or 2, which is a single chain antibody construct. [Aspect 4] The third domain is composed of, in order from amino to carboxyl: Hinge-CH2-CH3-Linker-Hinge-CH2-CH3 4. The bispecific antibody construct according to embodiment 2 or 3, comprising: [Aspect 5] 5. The bispecific antibody construct according to any one of aspects 1 to 4, wherein each of the polypeptide monomers in the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 24. [Aspect 6] 6. The bispecific antibody construct according to any one of aspects 1 to 5, wherein each of the polypeptide monomers has an amino acid sequence selected from SEQ ID NOs: 17 to 24. [Aspect 7] 7. The bispecific antibody construct according to any one of aspects 1 to 6, wherein the CH2 domain comprises an intradomain cysteine ​​disulfide bridge. [Aspect 8] (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) The bispecific antibody construct according to any one of aspects 1 to 7, wherein the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain. [Aspect 9] 9. The bispecific antibody construct according to any one of aspects 1 to 8, wherein the first domain and the second domain are fused to the third domain via a peptide linker. [Aspect 10] From amino to carboxyl, (a) the first domain; (b) a peptide linker preferably having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) the second domain 10. The bispecific antibody construct according to any one of embodiments 1 to 9, comprising: [Aspect 11] From amino to carboxyl, (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12; (e) a first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (g) a second polypeptide monomer of the third domain. 11. The bispecific antibody construct according to embodiment 10, further comprising: [Aspect 12] 12. The bispecific antibody construct according to any one of aspects 1 to 11, wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 528 (aa 4171 to 4296 according to uniprot Q685J3 numbering). [Aspect 13] 13. The bispecific antibody construct according to any one of aspects 1 to 12, wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 529 (aa 4189 to 4291 according to uniprot Q685J3 numbering). [Aspect 14] 14. The bispecific antibody construct according to any one of aspects 1 to 13, wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 530 (aa 4131-4243 according to uniprot Q685J3 numbering). [Aspect 15] 15. The bispecific antibody construct according to any one of aspects 1 to 14, wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 531 (aa 4244 to 4389 according to uniprot Q685J3 numbering). [Aspect 16] 16. The bispecific antibody construct according to any one of aspects 1 to 15, wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 530 (aa 4131-4243 according to uniprot Q685J3 numbering) but does not bind to an epitope in MUC17 corresponding to SEQ ID NO: 531 (aa 4244-4389 according to uniprot Q685J3 numbering). [Aspect 17] 17. The bispecific antibody construct according to any one of aspects 1 to 16, wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 532 (aas 4171-4390 according to uniprot Q685J3 numbering) or SEQ ID NO: 533 (aas 4184-4390 according to uniprot Q685J3 numbering) but does not bind to an epitope in MUC17 corresponding to SEQ ID NO: 534 (aas 4291-4390 according to uniprot Q685J3 numbering) or an epitope in MUC17 corresponding to SEQ ID NO: 535 (aas 4341-4390 according to uniprot Q685J3 numbering). [Aspect 18] The ratio between cytotoxicity and binding affinity (EC 50 / K D 18. The bispecific antibody construct according to any one of aspects 1 to 17, wherein R(x)*1000 is less than 250, said cytotoxicity being determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and said binding affinity being determined by a surface plasmon resonance based assay. [Aspect 19] The ratio between cytotoxicity and binding affinity (EC 50 / K D 19. The bispecific antibody construct according to any one of aspects 1 to 18, wherein R(x)*1000 is less than 125, said cytotoxicity being determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and said binding affinity being determined by a surface plasmon resonance based assay. [Aspect 20] The ratio between cytotoxicity and binding affinity (EC 50 / K D 20. The bispecific antibody construct according to any one of aspects 1 to 19, wherein R(x)*1000 is less than 21, said cytotoxicity being determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and said binding affinity being determined by a surface plasmon resonance based assay. [Aspect 21] The first binding domain comprises: (a) CDR-H1 as set forth in SEQ ID NO: 33, CDR-H2 as set forth in SEQ ID NO: 34, and CDR-H3 as set forth in SEQ ID NO: 35; (b) CDR-H1 as set forth in SEQ ID NO: 44, CDR-H2 as set forth in SEQ ID NO: 45, and CDR-H3 as set forth in SEQ ID NO: 46; (c) CDR-H1 as set forth in SEQ ID NO:55, CDR-H2 as set forth in SEQ ID NO:56, and CDR-H3 as set forth in SEQ ID NO:57; (d) CDR-H1 as set forth in SEQ ID NO:66, CDR-H2 as set forth in SEQ ID NO:67, and CDR-H3 as set forth in SEQ ID NO:68; (e) CDR-H1 as set forth in SEQ ID NO: 77, CDR-H2 as set forth in SEQ ID NO: 78, and CDR-H3 as set forth in SEQ ID NO: 79; (f) CDR-H1 as set forth in SEQ ID NO: 88, CDR-H2 as set forth in SEQ ID NO: 89, and CDR-H3 as set forth in SEQ ID NO: 90; (g) CDR-H1 as set forth in SEQ ID NO: 99, CDR-H2 as set forth in SEQ ID NO: 100, and CDR-H3 as set forth in SEQ ID NO: 101; (h) CDR-H1 as set forth in SEQ ID NO: 110, CDR-H2 as set forth in SEQ ID NO: 111, and CDR-H3 as set forth in SEQ ID NO: 112; (i) CDR-H1 as set forth in SEQ ID NO: 121, CDR-H2 as set forth in SEQ ID NO: 122, and CDR-H3 as set forth in SEQ ID NO: 123; (j) CDR-H1 as set forth in SEQ ID NO: 132, CDR-H2 as set forth in SEQ ID NO: 133, and CDR-H3 as set forth in SEQ ID NO: 134; (k) CDR-H1 as set forth in SEQ ID NO: 143, CDR-H2 as set forth in SEQ ID NO: 144, and CDR-H3 as set forth in SEQ ID NO: 145; (l) CDR-H1 as set forth in SEQ ID NO: 154, CDR-H2 as set forth in SEQ ID NO: 155, and CDR-H3 as set forth in SEQ ID NO: 156; (m) CDR-H1 as set forth in SEQ ID NO: 165, CDR-H2 as set forth in SEQ ID NO: 166, and CDR-H3 as set forth in SEQ ID NO: 167; (n) CDR-H1 as set forth in SEQ ID NO: 176, CDR-H2 as set forth in SEQ ID NO: 177, and CDR-H3 as set forth in SEQ ID NO: 178; (o) CDR-H1 as set forth in SEQ ID NO: 187, CDR-H2 as set forth in SEQ ID NO: 188, and CDR-H3 as set forth in SEQ ID NO: 189; (p) CDR-H1 as set forth in SEQ ID NO: 198, CDR-H2 as set forth in SEQ ID NO: 199, and CDR-H3 as set forth in SEQ ID NO: 200; (q) CDR-H1 as set forth in SEQ ID NO: 209, CDR-H2 as set forth in SEQ ID NO: 210, and CDR-H3 as set forth in SEQ ID NO: 211; (r) CDR-H1 as set forth in SEQ ID NO: 220, CDR-H2 as set forth in SEQ ID NO: 221, and CDR-H3 as set forth in SEQ ID NO: 222; (s) CDR-H1 as set forth in SEQ ID NO: 231, CDR-H2 as set forth in SEQ ID NO: 232, and CDR-H3 as set forth in SEQ ID NO: 233; (t) CDR-H1 as set forth in SEQ ID NO: 242, CDR-H2 as set forth in SEQ ID NO: 243, and CDR-H3 as set forth in SEQ ID NO: 244; (u) CDR-H1 as set forth in SEQ ID NO: 253, CDR-H2 as set forth in SEQ ID NO: 254, and CDR-H3 as set forth in SEQ ID NO: 255; (v) CDR-H1 as set forth in SEQ ID NO:264, CDR-H2 as set forth in SEQ ID NO:265, and CDR-H3 as set forth in SEQ ID NO:266; (w) CDR-H1 as set forth in SEQ ID NO: 275, CDR-H2 as set forth in SEQ ID NO: 276, and CDR-H3 as set forth in SEQ ID NO: 276; (x) CDR-H1 as set forth in SEQ ID NO: 286, CDR-H2 as set forth in SEQ ID NO: 287, and CDR-H3 as set forth in SEQ ID NO: 288; (y) CDR-H1 as set forth in SEQ ID NO:297, CDR-H2 as set forth in SEQ ID NO:298, and CDR-H3 as set forth in SEQ ID NO:299; (z) CDR-H1 as set forth in SEQ ID NO: 308, CDR-H2 as set forth in SEQ ID NO: 309, and CDR-H3 as set forth in SEQ ID NO: 310; (aa) CDR-H1 as set forth in SEQ ID NO: 319, CDR-H2 as set forth in SEQ ID NO: 320, and CDR-H3 as set forth in SEQ ID NO: 321; (ab) CDR-H1 as set forth in SEQ ID NO: 330, CDR-H2 as set forth in SEQ ID NO: 331, and CDR-H3 as set forth in SEQ ID NO: 332; (ac) CDR-H1 as set forth in SEQ ID NO: 341, CDR-H2 as set forth in SEQ ID NO: 342, and CDR-H3 as set forth in SEQ ID NO: 343; (ad) CDR-H1 as set forth in SEQ ID NO: 352, CDR-H2 as set forth in SEQ ID NO: 353, and CDR-H3 as set forth in SEQ ID NO: 354; (ae) CDR-H1 as set forth in SEQ ID NO: 363, CDR-H2 as set forth in SEQ ID NO: 364, and CDR-H3 as set forth in SEQ ID NO: 365; (af) CDR-H1 as set forth in SEQ ID NO: 374, CDR-H2 as set forth in SEQ ID NO: 375, and CDR-H3 as set forth in SEQ ID NO: 376; (ag) CDR-H1 as set forth in SEQ ID NO: 385, CDR-H2 as set forth in SEQ ID NO: 386, and CDR-H3 as set forth in SEQ ID NO: 386; (ah) CDR-H1 as set forth in SEQ ID NO: 396, CDR-H2 as set forth in SEQ ID NO: 397, and CDR-H3 as set forth in SEQ ID NO: 398; (ai) CDR-H1 as set forth in SEQ ID NO: 407, CDR-H2 as set forth in SEQ ID NO: 408, and CDR-H3 as set forth in SEQ ID NO: 409; (aj) CDR-H1 as set forth in SEQ ID NO: 418, CDR-H2 as set forth in SEQ ID NO: 419, and CDR-H3 as set forth in SEQ ID NO: 420; (ak) CDR-H1 as set forth in SEQ ID NO: 429, CDR-H2 as set forth in SEQ ID NO: 430, and CDR-H3 as set forth in SEQ ID NO: 431; (a1) CDR-H1 as set forth in SEQ ID NO: 440, CDR-H2 as set forth in SEQ ID NO: 441, and CDR-H3 as set forth in SEQ ID NO: 442; (am) CDR-H1 as set forth in SEQ ID NO: 451, CDR-H2 as set forth in SEQ ID NO: 452, and CDR-H3 as set forth in SEQ ID NO: 453; (an) CDR-H1 as set forth in SEQ ID NO: 462, CDR-H2 as set forth in SEQ ID NO: 463, and CDR-H3 as set forth in SEQ ID NO: 464; (ao) CDR-H1 as set forth in SEQ ID NO: 473, CDR-H2 as set forth in SEQ ID NO: 474, and CDR-H3 as set forth in SEQ ID NO: 475; (ap) CDR-H1 as set forth in SEQ ID NO: 484, CDR-H2 as set forth in SEQ ID NO: 485, and CDR-H3 as set forth in SEQ ID NO: 486; (aq) CDR-H1 as set forth in SEQ ID NO: 495, CDR-H2 as set forth in SEQ ID NO: 496, and CDR-H3 as set forth in SEQ ID NO: 497; (ar) CDR-H1 as set forth in SEQ ID NO: 506, CDR-H2 as set forth in SEQ ID NO: 507, and CDR-H3 as set forth in SEQ ID NO: 508; and (as) CDR-H1 as set forth in SEQ ID NO: 517, CDR-H2 as set forth in SEQ ID NO: 518, and CDR-H3 as set forth in SEQ ID NO: 519. a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from: (c) CDR-H1 as set forth in SEQ ID NO:55, CDR-H2 as set forth in SEQ ID NO:56, and CDR-H3 as set forth in SEQ ID NO:57; (n) CDR-H1 as set forth in SEQ ID NO: 176, CDR-H2 as set forth in SEQ ID NO: 177, and CDR-H3 as set forth in SEQ ID NO: 178; (ac) CDR-H1 as set forth in SEQ ID NO: 341, CDR-H2 as set forth in SEQ ID NO: 342, and CDR-H3 as set forth in SEQ ID NO: 343; and (aj) CDR-H1 as set forth in SEQ ID NO: 418, CDR-H2 as set forth in SEQ ID NO: 419, and CDR-H3 as set forth in SEQ ID NO: 420 The bispecific antibody construct according to any one of aspects 1 to 20, wherein [Aspect 22] The first binding domain comprises: (a) CDR-L1 as set forth in SEQ ID NO: 36, CDR-L2 as set forth in SEQ ID NO: 37, and CDR-L3 as set forth in SEQ ID NO: 38; (b) CDR-L1 as set forth in SEQ ID NO: 47, CDR-L2 as set forth in SEQ ID NO: 48, and CDR-L3 as set forth in SEQ ID NO: 49; (c) CDR-L1 as set forth in SEQ ID NO:58, CDR-L2 as set forth in SEQ ID NO:59, and CDR-L3 as set forth in SEQ ID NO:60; (d) CDR-L1 as set forth in SEQ ID NO:69, CDR-L2 as set forth in SEQ ID NO:70, and CDR-L3 as set forth in SEQ ID NO:71; (e) CDR-L1 as set forth in SEQ ID NO: 80, CDR-L2 as set forth in SEQ ID NO: 81, and CDR-L3 as set forth in SEQ ID NO: 82; (f) CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93; (g) CDR-L1 as set forth in SEQ ID NO: 102, CDR-L2 as set forth in SEQ ID NO: 103, and CDR-L3 as set forth in SEQ ID NO: 104; (h) CDR-L1 as set forth in SEQ ID NO: 113, CDR-L2 as set forth in SEQ ID NO: 114, and CDR-L3 as set forth in SEQ ID NO: 115; (i) CDR-L1 as set forth in SEQ ID NO: 124, CDR-L2 as set forth in SEQ ID NO: 125, and CDR-L3 as set forth in SEQ ID NO: 126; (j) CDR-L1 as set forth in SEQ ID NO: 135, CDR-L2 as set forth in SEQ ID NO: 136, and CDR-L3 as set forth in SEQ ID NO: 137; (k) CDR-L1 as set forth in SEQ ID NO: 146, CDR-L2 as set forth in SEQ ID NO: 147, and CDR-L3 as set forth in SEQ ID NO: 148; (l) CDR-L1 as set forth in SEQ ID NO: 157, CDR-L2 as set forth in SEQ ID NO: 158, and CDR-L3 as set forth in SEQ ID NO: 159; (m) CDR-L1 as set forth in SEQ ID NO: 168, CDR-L2 as set forth in SEQ ID NO: 169, and CDR-L3 as set forth in SEQ ID NO: 170; (n) CDR-L1 as set forth in SEQ ID NO: 179, CDR-L2 as set forth in SEQ ID NO: 180, and CDR-L3 as set forth in SEQ ID NO: 181; (o) CDR-L1 as set forth in SEQ ID NO: 190, CDR-L2 as set forth in SEQ ID NO: 191, and CDR-L3 as set forth...

Claims

1. 1. A bispecific antibody construct comprising: a first domain that binds to MUC17, and A second domain that binds to an extracellular epitope of the human and macaque CD3 epsilon chain Including, Here, The first domain comprises a VH region comprising CDR-H1 as set forth in SEQ ID NO:55, CDR-H2 as set forth in SEQ ID NO:56, and CDR-H3 as set forth in SEQ ID NO:57, and the first domain comprises an LH region comprising CDR-L1 as set forth in SEQ ID NO:58, CDR-L2 as set forth in SEQ ID NO:59, and CDR-L3 as set forth in SEQ ID NO:60; The first domain comprises a VH region comprising CDR-H1 as set forth in SEQ ID NO:418, CDR-H2 as set forth in SEQ ID NO:419, and CDR-H3 as set forth in SEQ ID NO:420, and the first domain comprises an LH region comprising CDR-L1 as set forth in SEQ ID NO:421, CDR-L2 as set forth in SEQ ID NO:422, and CDR-L3 as set forth in SEQ ID NO:423; or The first domain comprises a VH region comprising CDR-H1 as set forth in SEQ ID NO:473, CDR-H2 as set forth in SEQ ID NO:474, and CDR-H3 as set forth in SEQ ID NO:475, and the first domain comprises an LH region comprising CDR-L1 as set forth in SEQ ID NO:476, CDR-L2 as set forth in SEQ ID NO:477, and CDR-L3 as set forth in SEQ ID NO:478; The bispecific antibody construct.

2. 2. The bispecific antibody construct of claim 1, further comprising a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain, said two polypeptide monomers being fused to each other via a peptide linker.

3. 3. The bispecific antibody construct of claim 1 or 2, which is a single chain antibody construct.

4. The third domain is composed of, in order from amino to carboxyl: Hinge-CH2-CH3-linker-hinge-CH2-CH3 3. The bispecific antibody construct of claim 2, comprising:

5. 5. The bispecific antibody construct of claim 2 or 4, wherein each of the polypeptide monomers in the third domain has an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-24.

6. 6. The bispecific antibody construct of any one of claims 2, 4 to 5, wherein each of said polypeptide monomers has an amino acid sequence selected from SEQ ID NOs: 17 to 24.

7. 7. The bispecific antibody construct of claim 2, wherein the CH2 domain comprises an intradomain cysteine ​​disulfide bridge.

8. (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain.

9. 8. The bispecific antibody construct of any one of claims 2, 4 to 7, wherein the first and second domains are fused to the third domain via a peptide linker.

10. From amino to carboxyl, (a) the first domain; (b) a peptide linker; and (c) the second domain 10. The bispecific antibody construct according to claim 1 , comprising:

11. From amino to carboxyl, (a) the first domain; (b) a peptide linker; (c) the second domain; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12; (e) a first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (g) a second polypeptide monomer of the third domain.

10. The bispecific antibody construct of claims 2, 4 to 7, or 9, further comprising:

12. The bispecific antibody construct according to claim 10 or 11, wherein the (b) peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

13. The first domain may be one of the following: (c) a VL region as set forth in SEQ ID NO:62 and a VH region as set forth in SEQ ID NO:61; (aj) a VL region as set forth in SEQ ID NO:425 and a VH region as set forth in SEQ ID NO:424; (ao) a VL region as set forth in SEQ ID NO: 480 and a VH region as set forth in SEQ ID NO: 479; comprising a VL region and a VH region selected from the group consisting of: A bispecific antibody construct according to any one of claims 1 to 12.

14. 14. The bispecific antibody construct according to any one of claims 1 to 13, comprising a sequence selected from the amino acid sequences of SEQ ID NOs: 63, 426 and 481.

15. An amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 427, 428, 482 or 483; or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the amino acid sequence 15. The bispecific antibody construct according to any one of claims 1 to 14, comprising:

16. A polynucleotide encoding an antibody construct described in any one of claims 1 to 15.

17. A vector comprising the polynucleotide described in claim 16.

18. A host cell transformed or transfected with the polynucleotide of claim 16 or the vector of claim 17.

19. A method for producing a bispecific antibody construct according to any one of claims 1 to 15, comprising culturing a host cell according to claim 18 under conditions allowing expression of an antibody construct according to any one of claims 1 to 15, and recovering the expressed antibody construct from the culture.

20. A pharmaceutical composition comprising a bispecific antibody construct according to any one of claims 1 to 15.

21. 21. The pharmaceutical composition of claim 20, which is stable at -20°C for at least 4 weeks.

22. 21. The pharmaceutical composition according to claim 20 for use in the prevention, treatment or amelioration of a disease selected from a proliferative disease, a neoplastic disease, a cancer or an immune disorder.

23. 23. The pharmaceutical composition of claim 22, wherein the disease is gastrointestinal cancer or pancreatic cancer.

24. The pharmaceutical composition of claim 22, wherein the disease is gastric cancer.

25. A kit comprising the bispecific antibody construct of any one of claims 1 to 15, the polynucleotide of claim 16, the vector of claim 17, and / or the host cell of claim 18.

26. A pharmaceutical composition for the treatment or amelioration of gastrointestinal cancer comprising a bispecific antibody construct according to any one of claims 1 to 15, which is directed against MUC17 and CD3.

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