Bispecific antibody constructs directed against MUC17 and CD3

Abstract

The present invention provides bispecific antibody constructs characterized by comprising a 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 that is in a specific Fc format. The present invention also provides polynucleotides encoding the antibody constructs, vectors containing the polynucleotides, host cells that express the constructs, and pharmaceutical compositions comprising the same.

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 developing therapeutics are protein-based drugs, which have already played important roles in nearly 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 to small molecule chemical drugs, protein drugs have high specificity and activity at relatively low concentrations, typically providing treatment for high-impact diseases 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 drugs include, for example, bispecific (monoclonal) antibodies, which are typically capable of simultaneously binding two different types of antigens. They are known in several structural forms 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 typically retain the typical monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab regions bind to different antigens. Non-full-length bispecific antibodies may completely lack the Fc region. These include chemically linked Fabs consisting of only the Fab region and various types of bivalent and trivalent single-chain variable fragments (scFvs). 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 a BiTE® antibody construct is specific for a selected tumor-associated surface antigen on a target cell, and the second binding domain is specific for CD3, a subunit of the T cell receptor complex on a T cell. Due to their special design, BiTE® antibody constructs are uniquely suited to transiently link T cells to target cells while simultaneously potently activating the intrinsic cytolytic potential of T cells against the target cells. An important further development of the first generation BiTE® antibody constructs deployed in the clinic as AMG103 and AMG110 (see WO 99 / 54440 and WO 2005 / 040220) provided bispecific antibody constructs that bind to a context-independent epitope at the N-terminus of the CD3 epsilon chain (WO 2008 / 119567). BiTE® antibody constructs that bind to this selected epitope not only lack cross-species specificity for human and macaque (Macaca) or marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), or squirrel monkey (Saimiri sciureus) CD3ε chains, but also, because they recognize this specific epitope (instead of the CD3-binding epitope previously described in bispecific T cell-engaging molecules), do not exhibit the same degree of nonspecific activation of T cells 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 has been considered a risk factor for 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 rapidly reach most parts of the body, their in vivo application may be limited by their short in vivo persistence. On the other hand, their concentration in the body can be readily adapted and fine-tuned. Because of the short in vivo half-life of these small single-chain molecules, continuous administration by continuous intravenous infusion is used to achieve therapeutic effects. However, novel bispecific antibody constructs are available that have more advantageous pharmacokinetic properties, including longer half-lives. A long half-life is generally useful in the in vivo application of immunoglobulins, particularly antibodies, and most particularly small-sized antibody fragments or constructs, 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, which 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 the regulation of proliferation and apoptosis, and tumorigenesis (Hollingsworth and Swanson, Nat. Rev. Cancer 2004). Among mucins, mucin 17 (MUC17) was 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, an epidermal growth factor (EGF) domain, a sea urchin sperm protein, enterokinase, and agrin (SEA) domain, and a large extracellular domain composed of a second EGF domain. The SEA domain contains a putative cleavage site conserved in other mucins (Moniaux et al., J. Biol. Chem. 2006). MUC17 is a single-pass transmembrane protein with an intracellular 80-amino acid cytoplasmic tail (Moniaux et al., J. Biol. Chem. 2006). In healthy adults, MUC17 expression 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. Immunology 2016). 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 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 cell lines (Gum et al. 2002). Immunohistochemistry confirmed MUC17 protein expression in pancreatic cancer (Moniaux et al. 2006). However, MUC17 protein expression was shown to be reduced in colon cancer (Senapati et al., 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] Given the conflicting views in the literature regarding MUC17 as a potential target for certain pathological conditions, it is an object of the present invention to provide bispecific antibody constructs, such as T cell engaging molecules, that clearly identify specific conditions associated with MUC17 upregulation and are particularly suitable for binding MUC17 in MUC17-associated conditions, preferably for use in treating said specific conditions. Accordingly, the present invention provides antibody constructs characterized by comprising a first domain that binds to MUC17, a second domain that binds to an extracellular epitope of human and non-human, e.g., macaque, CD3 epsilon chains, and a third domain, preferably in a specific Fc format. Furthermore, the present invention provides polynucleotides encoding the antibody constructs, vectors containing the polynucleotides, and host cells expressing the constructs, as well as pharmaceutical compositions comprising the same. [Means for solving the problem]

[0011] In a first aspect, in the context of the present invention, a first domain that binds to MUC17, and A second domain that binds to extracellular epitopes of human and macaque CD3ε chains 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 the context of 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 envisioned in relation to the present invention that an antibody construct is provided, in which each of the polypeptide monomers has an amino acid sequence selected from SEQ ID NOs: 17 to 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) 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 amino groups in the 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) the 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 contemplated that the present invention provides an antibody construct, wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 528 (aa 4171-4296 according to Uniprot Q685J3 numbering).

[0021] In the above aspect, it is also contemplated that the present invention provides 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 aspect, it is further contemplated that the present invention provides an antibody construct, wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 530 (aa 4131-4243 according to Uniprot Q685J3 numbering).

[0023] In the above aspect, it is also contemplated that the present invention provides 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 contemplated that the present invention provides an antibody construct wherein the first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 530 (aa 4131-4243 according to uniprot Q685J3 numbering) but does not bind to an epitope within MUC17 corresponding to SEQ ID NO: 531 (aa 4244-4389 according to uniprot Q685J3 numbering).

[0025] In the above aspects, it is also contemplated that the present invention provides an antibody construct wherein the first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO: 532 (aa 4171-4390 according to uniprot Q685J3 numbering) or SEQ ID NO: 533 (aa 4184-4390 according to uniprot Q685J3 numbering), but does not bind to an epitope in MUC17 corresponding to SEQ ID NO: 534 (aa 4291-4390 according to uniprot Q685J3 numbering) or an epitope in MUC17 corresponding to SEQ ID NO: 535 (aa 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 )*1000 is less than 250, the cytotoxicity is expressed in pM and is determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and the binding affinity is expressed in nM and is determined by a surface plasmon resonance (SPR) assay, such as a Biacore assay. 50 value and K D The factor 1000 is introduced to improve readability and account for 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, the cytotoxicity is given in pM and is determined, for example, in NUGC-4 cells as target cells and huPBMCs as effector cells, and the 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 )*1000 is less than 21, the cytotoxicity is given in pM and is determined, for example, in NUGC-4 cells as target cells and huPBMCs as effector cells, and the binding affinity is given in nM and is determined by a surface plasmon resonance based assay. Preferably, the cytotoxicity (EC 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, 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 It is also envisaged to provide an antibody construct, wherein:

[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, CDR-L2 as set forth in SEQ ID NO: 290, 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 It is also envisaged to provide an antibody construct, wherein:

[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 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 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 further envisaged in the context of the present invention to provide an antibody construct, wherein the antibody construct comprises a sequence selected from the amino acid sequences as set forth in any 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.

[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 amino groups in the 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 the 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 aspect, 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, 308, 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,

[0037] It is also contemplated to provide antibody constructs 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 an antibody construct of the present invention. In a third aspect, the present invention also envisages providing a vector comprising a polynucleotide of the present 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 a polynucleotide or a 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 present invention or an antibody construct produced according to a method of the present 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 the context of 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, the method comprising 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 cancer or pancreatic cancer, most preferably gastric cancer.

[0043] In an eighth aspect, it is also envisaged in the context of 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 explanation of the drawings]

[0046] [Figure 1] Epitope clustering of MUC17 is shown. Epitopes E1, E2, E3, E4, E5A, and E5B, as well as truncated forms of E2 (TR2, TR3, TR4, and TR5, respectively), are marked. Experiments with constructs in which human MUC17 (brown / gray) was replaced with non-functional mouse MUC3 revealed the respective epitopes. 45 MUC-17-scFc bispecific antibody constructs were identified that spanned the epitope space E2, including the SEA domain. [Figure 2] Figure 1 shows 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 MUC17-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. [Figure 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 a 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 were: 1 = 32-G6; 2 = 1-B6; 3 = 2-C2; and 4 = 8-B7. Construct 8-B7 showed a slight advantage in terms of cytotoxicity. [Figure 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 hours of incubation (target cells GSU (A) or NUGC-4 (B), 10:1 human T cells to target cells, SteadyGlo readout). Addition of sMUC17 did not affect the cytotoxic activity of the bispecific antibody construct. [Figure 6] The MUC17-scFc antibody construct 8-B7 inhibits tumor growth in a xenograft model of colorectal cancer. Female NOD / SCID mice were implanted with 2 x 10 Ls174T colorectal cancer cells. On day 15, activated T cells expanded to 2 x 10 were administered by intraperitoneal (IP) injection. The 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 in NUGC-4 cells (EC50) in [pM], the ratio (EC50 / KD)*1000, and VH VL configuration. [Figure 8] The 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 of MUC17 HLE BiTE® at 0 and 168 hours. Serum was collected at the indicated time points and analyzed for the presence of the MUC17 scFc bispecific antibody construct using ELISAs based on either anti-CD3 or anti-MUC17 antibodies. Data were fit to a two-compartment model. Graphs show individual data (points) and mean values ​​(lines). DETAILED DESCRIPTION OF THE INVENTION

[0047] In accordance with the present invention, a bispecific antibody construct is provided that specifically targets MUC17, which is associated with malignant tumors. To this end, MUC17 was first identified as a gene upregulated in gastric tumors relative to normal tissue expression. In this regard, MUC17 protein has been shown to be expressed in 40-77% of gastric tumors using immunohistochemistry methods commonly used in the art. Flow cytometry has also demonstrated that MUC17 protein is expressed on the cell surface of gastric and esophageal cancer cell lines, as well as some pancreatic and colorectal cancer cell lines. Such expression has also been shown to be particularly high in gastric tumors in Chinese patients. Therefore, MUC17 is identified as a valid target associated with gastrointestinal cancer, namely, gastric, small intestinal, and large intestinal (colon) cancer, esophageal cancer, and pancreatic cancer.

[0048] In the context of the present invention, it is a surprising discovery that bispecific antibody constructs according to the present invention preferably target cancer cells, such as gastric and gastrointestinal cancer cells, bearing MUC17, and, in contrast, target fewer non-cancerous cells. MUC17 is typically expressed on the apical surface of non-cancerous intestinal epithelial cells (i.e., located opposite the basal surface of each cell) and forms part of the mucosal layer. However, MUC17 is overexpressed in gastric and gastrointestinal cancers, and in such situations, it 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 bispecific antibody constructs according to the present invention, whereas MUC17 expressed on non-mucosal surfaces in cancer cells is more accessible. Thus, bispecific antibody constructs according to the present invention preferably target MUC17-associated cancer cells and target fewer non-cancerous cells. This was surprisingly found when comparing good tolerability in healthy animals with 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, although advantageously, there were no histopathological changes in the MUC17-expressing tissues. The good tolerability of bispecific antibody constructs according to the invention by non-cancerous cells is likewise confirmed in vitro. In contrast, intravenous treatment of tumor-bearing mice with bispecific antibody constructs according to the invention results in statistically significant and dose-dependent inhibition of tumor growth compared to placebo-treated mice in the control group. Thus, 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] The present invention provides a bispecific antibody construct directed against the EGF-SEA-EGF region of the MUC17 protein. Advantageously, targeting this region of the protein confers selectivity from its 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. Like other transmembrane mucins, MUC17 contains a potential cleavage site within the SEA domain.

[0050] Thus, bispecific antibody constructs having a single-chain Fc format that target the MUC17 EGF-SEA-EGF region and CD3 and that extend half-life targeting are contemplated herewith. Advantageously, the bispecific antibody constructs of the present 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 cross-reactive with, for example, cynomolgus MUC17 (in addition to human MUC17), enabling non-clinical toxicology testing. The importance of the details of the EGF-SEA-EGF domain sequence of cynomolgus MUC17 is demonstrated for the first time herein.

[0052] In the context of the present invention, bispecific antibody constructs are envisaged to exhibit binding affinity, potent cytotoxic activity and be the most stable maps to the SEA domain. In the context of the present invention, it is envisaged that the bispecific antibody construct will have a cysteine ​​clamp, i.e. an intramolecular disulfide bond, in the target binding moiety 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 extending (HLE) moiety and directed against MUC17 are envisaged for use in the treatment of gastrointestinal cancers, including gastric cancer, gastroesophageal cancer, esophageal cancer, pancreatic cancer 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 were first identified by removing the tandem repeats of MUC17 because they are highly glycosylated and repetitive in sequence. This resulted in, for example, the underlined region at 376 aa and the EGF-like / SEA domain region at 177 aa. Advantageously, targeting the EGF-like / SEA domain allows for selectivity from closest family members such as MUC3, cross-reactivity with cynomolgus MUC17, and binding to membrane-bound MUC17. Subsequently, the inventors developed reagents and assays to assess binding and T cell redirection for 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 epitopes of preferred bispecific antibody constructs directed against MUC17, epitope mapping was performed as described herein. Preferred bispecific antibody constructs are 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, which essentially corresponds to aa 4171-4296 of MUC17 according to the uniprot Q685J3 numbering. Generally, in the context of the present invention, the aa numbering of MUC17 is always generated or intended to be generated with reference to the uniprot Q685J3 numbering of MUC17. Meanwhile, bispecific antibody constructs targeting the E1 epitope of MUC17, i.e., the epitope N-terminal to the SEA domain (see FIG. 1 ), surprisingly exhibit undesirable cross-reactivity with MUC3A and MUC3B, resulting in off-target activity and ultimately an increased risk of side effects. Furthermore, bispecific antibody constructs directed against epitopes E3 and E4 located at the C-terminus of the SEA domain (see Figure 1) unexpectedly do not cross-react with cynomolgus MUC17. It is therefore envisioned that bispecific antibody constructs according to the invention specifically and exclusively bind to the E2 epitope of MUC17.

[0057] Such preferred bispecific antibody constructs according to the present invention can be further identified based on their structure or their unique detailed epitope binding properties. Preferred bispecific antibody constructs according to the present invention can be determined by calculating a novel indicative ratio of cytotoxicity and affinity as provided herein. For example, the 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), while a ratio >250 usually indicates good binding to TR2 but not to TR3. In particular, the most preferred constructs usually bind to part of the epitope cluster E2 / E5A / 5B and / or TR2 / TR3. They are, for example, less than about 21 (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 identified in the context of the present invention as, 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. The constructs bind to part of the epitope cluster E2 / E5A / 5B and / or TR2 / TR3 and have an EC of less than about 125. 50 :K D Also preferred are constructs that exhibit a ratio and belong to sequence families (OPT library designations) 1a, 1c, and 9. Their VH / VL configurations are characterized as 3lambda3 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 average of less than about 1500, typically 250-1450 (EC 50 / K DBinders exhibiting a )*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 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 typically measured by SPC, such as Biacore B analysis, with results typically given in nM. Cytotoxic activity is typically 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 envisioned that preferred bispecific antibody constructs not only exhibit a favorable ratio of cytotoxicity to affinity, but also exhibit sufficient stability characteristics to facilitate practical handling during formulation, storage, and administration of the construct. Sufficient stability is characterized 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). Furthermore, the turbidity measured as light absorbance 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, for example, an essential absence of undesired aggregates. Advantageously, the high monomer content is maintained after incubation under stress conditions, such as freeze / thaw, or at 37 or 40°C.

[0059] Therefore, 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 CD3ε chain; and optionally, a third domain comprising two polypeptide monomers, each comprising 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 present invention is herein understood as a binding domain of an antibody construct according to the present invention. Typically, a binding domain according to the present invention comprises the minimum structural requirements of an antibody that enable target binding. This minimum requirement may be defined, for example, 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 by defining the epitope of the antibody within the structure of a specific target, protein domains of the target protein (epitope clusters) that each constitute an epitope region, or by reference to specific antibodies that compete 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 an 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 comprise both. An Fd fragment, for example, has two VH regions and often retains some of the antigen-binding function of an 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) an 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, which has two VH and CH1 domains; (4) an Fv fragment, which has the VL and VH domains of one arm of an antibody; (5) a dAb fragment, which has 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 is preferred (e.g., derived 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 Patent Application Publication No. 2014 / 0308285, US Patent Application Publication No. 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, which comprise 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 contain a polypeptide linker between the VH and VL domains that enables them to form the desired structure that enables antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monoclonal Antibodies," vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods for making single chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. 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, and therefore 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 are described, inter alia, in Harlow and Lane, Antibodies 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, wherein 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 present invention has 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 expressed by a cell and present on its cell surface so as to be accessible to the antibody constructs described herein. It may be a protein, preferably the 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 antibody constructs according to the present invention are (at least) bispecific, they do not occur in nature and are significantly different 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. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0067] The at least two binding domains and variable domain (VH / VL) of the antibody construct of the present invention may or may not contain a peptide linker (spacer peptide). According to the present invention, the term "peptide linker" includes an amino acid sequence that interconnects the amino acid sequences of one (variable and / or binding) domain and another (variable and / or binding) domain of the antibody construct of the present invention. A peptide linker can also be used to fuse a third domain to another domain of the antibody construct of the present invention. The essential technical feature of such a peptide linker is that it does not contain any polymerization activity. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344. A peptide linker can also be used to link other domains, 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 as defined above in which all or a part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection, such as in vitro phage display, protein chip, or any other method that allows testing of candidate sequences for antigen-binding ability. Thus, this term preferably excludes sequences generated solely by genome rearrangement in immune cells of an animal. A "recombinant antibody" is an antibody generated by using 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), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a 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 cultures can be used. For example, the monoclonal antibodies used can be made by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by recombinant DNA methods (see, e.g., 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 antibodies that specifically bind to the designated antigen. Any form of the relevant antigen can be used as the immunogen, including, for example, recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof. Surface plasmon resonance, as employed in the Biacore system, can be used to increase the efficiency of phage antibody binding 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 using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (such as mice, hamsters, rabbits, or rats). In one embodiment, the non-human animal contains 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, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0074] Monoclonal antibodies can also be obtained from non-human animals and then 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 modified effector function (see, e.g., U.S. Pat. No. 5,648,260; Kontermann and Duebel (2010), supra; and Little (2009), supra).

[0075] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for an antigen during an immune response. Repeated exposure to the same antigen leads the host to produce antibodies with successively increasing affinities. Similar to natural prototyping, 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 CDRs can be introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinities in the low nanomolar range.

[0076] A preferred type of amino acid substitution variant of an antibody construct involves substituting 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 were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, 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 using the techniques detailed herein. After generating such variants, the panel of variants can be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0077] The monoclonal antibodies and antibody constructs of the present invention particularly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to 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 to 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 the desired biological activity is exhibited (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 comprising 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 can also be modified by specific deletion of human T-cell epitopes (a method called "deimmunization") by methods disclosed in the Examples of WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of an 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). To detect potential T-cell epitopes, a computer modeling method called "peptide threading" can be applied, as described in WO 98 / 52976 and WO 00 / 34317. In addition, databases of human MHC class II-binding peptides can be searched for motifs present in 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 within 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 within human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in 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 VBASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (edited by Tomlinson, L.A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for framework regions and CDRs.For example, the consensus human framework regions described in US Pat. No. 6,300,064 can be used.

[0079] "Humanized" antibodies, antibody constructs, variants, or fragments thereof (Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins of largely human sequence that contain 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 called 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 either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. Humanized antibodies 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 involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of immunoglobulin Fv variable domains 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 incapable of expressing 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 some of the CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for the binding of the humanized antibody to a predetermined 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 (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982, and EP 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. Human antibodies, antibody constructs, or binding domains 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 technologies 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 constructs of the present invention are "isolated" or "substantially pure" antibody constructs. 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 from 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. An antibody construct may, for example, constitute at least about 5% by weight or at least about 50% by weight of the total protein in a given sample. It is understood that an isolated protein may constitute 5% to 99.9% by weight of the total protein content, depending on the circumstances. The use of inducible promoters or high-expression promoters to produce the polypeptide at high concentration levels can allow for production of significantly higher concentrations of the polypeptide. This definition includes production of antibody constructs in a variety of organisms and / or host cells known in the art. In preferred embodiments, the antibody construct will be purified (1) sufficiently 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 using 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), herein 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) 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 also comprises the minimal structural requirements of an antibody that enable target binding. 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 may be 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, a binding domain is in the form of one or more polypeptides. Such polypeptides may comprise 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" generally refers to a group of molecules consisting 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, e.g., modified 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 by 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 comprising 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 those derived from rodents (e.g., mouse, rat, hamster, or rabbit). The presence of such rodent-derived proteins can result in rapid clearance of the antibody or antibody construct or can lead to 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 function into rodents such that the rodents produce 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 loosely 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 insights 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. Furthermore, such a strategy would provide an ideal source for the generation of fully human monoclonal antibodies (mAbs), a key milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or antibody constructs are expected to minimize the immunogenicity and allergic reactions inherent in mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of administered antibodies / antibody constructs. The use of fully human antibodies or antibody constructs is expected to offer significant advantages in the treatment of chronic and recurrent human diseases that require repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0091] One approach toward 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. Large human Ig fragments would preserve the broad diversity of variable genes and the appropriate 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 desired specificity could be readily generated and selected. This general strategy was demonstrated in connection 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 germline-configured fragments of the human heavy chain and kappa light chain loci, respectively, that contained the core sequences of the variable and constant regions. These 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 generate 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 characteristic of 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 chain and kappa light chain loci 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 is 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 / 464,582, 08 / 463,191, 08 / 464,583, 08 / 464,584 ...4, 08 / 464,584, 08 / 464,584, 08 / 464,58 and 6,162,963; 6,150,584; 6,114,598; 6,075,181; and 5,939,598, and Japanese Patent Publications Nos. 3068180B2, 3068506B2, and 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310, and WO 03 / 47336.

[0093] In another approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In this 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. Patent Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns, U.S. Patent Nos. 5,612,205; 5,721,367; and 5,789,215 to Berns et al., and U.S. Patent 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 fragments or entire chromosomes have been introduced by microcell fusion. See European Patent Applications 773288 and 843961. Xenerex Biosciences is developing a promising human antibody production technology in which SCID mice are reconstituted with human lymphocytes, e.g., B cells and / or T cells. The mice are then immunized with an antigen and are able to generate an immune response against that antigen. See U.S. Patent Nos. 5,476,996; 5,698,767; and 5,958,765.

[0095] Human anti-mouse antibody (HAMA) responses have led the industry to generate chimeric or otherwise humanized antibodies. However, it is expected that some human anti-chimeric antibody (HACA) responses will be observed, particularly with chronic or multi-dose antibody use. Therefore, it would be desirable to provide an antibody construct comprising a human binding domain for MUC17 and a human binding domain for CD3ε to eliminate the concerns and / or impact of HAMA or HACA responses.

[0096] The terms "(specifically) bind", "(specifically) recognize", "(specifically) be attracted to" 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 therefore the term epitope is also sometimes referred to herein as an "antigenic structure" or "antigenic determinant." The binding domain is therefore an "antigen interaction site." It is understood that said binding / interaction also defines "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 recognized by a primary amino acid sequence. Linear epitopes typically contain at least three or at least four, and more usually at least five, or at least six, or at least seven, e.g., about 8 to about 10, amino acids in a unique sequence.

[0099] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary sequence of amino acids comprising the epitope is not the only defining element of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by a binding domain). Generally, conformational epitopes contain a larger number of amino acids than linear epitopes. In recognizing 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 forming the conformational epitope are juxtaposed, thereby enabling 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, site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0100] Epitope mapping methods are described below. When a region (a contiguous stretch of amino acids) of the human MUC17 protein is exchanged or substituted with the corresponding region of nonhuman and nonprimate MUC17 (e.g., mouse MUC17, but also chicken, rat, hamster, rabbit, etc.), a reduction in binding activity of the binding domain is expected, as long as the binding domain is not cross-reactive with the nonhuman, nonprimate MUC17 used. This 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 binding to the corresponding region in the human MUC17 protein, assuming binding to the corresponding region in the human MUC17 protein as 100%. It is envisioned that the above-described human MUC17 / non-human MUC17 chimeras will be expressed in CHO cells. It is also envisioned that human MUC17 / non-human MUC17 chimeras may be 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 generated to determine the specific region 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 contemplated that truncated MUC17 can be expressed in CHO cells. It is also contemplated that truncated MUC17 may be fused to the transmembrane and / or cytoplasmic domains of different membrane-associated proteins, such as EpCAM. It is also contemplated that truncated MUC17 may include a signal peptide domain at their N-terminus, such as a signal peptide derived from the mouse IgG heavy chain signal peptide. Furthermore, it is contemplated that truncated MUC17 may include a v5 domain at the N-terminus (following the signal peptide), which can confirm their correct expression on the cell surface. It is expected that truncated MUC17 forms that no longer include the MUC17 region recognized by the binding domain will exhibit reduced or lost binding. 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 whole 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, in which each residue being analyzed is substituted with alanine, for example, by site-directed mutagenesis (see, e.g., Morrison KL & Weiss GA. Cur Opin Chem Biol. 2001 Jun;5(3):302-7). Alanine is used because it is not bulky, is chemically inert, and yet contains a methyl functional group that mimics the secondary structure criteria of many other amino acids. If it is desirable to preserve the size of the residue being mutated, bulky amino acids such as valine or leucine can sometimes 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 epitope-containing region means that the binding domain exhibits measurable affinity for the epitope / epitope-containing region on a particular protein or antigen (MUC17 and CD3, respectively, herein), 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 Binding is considered specific when M is M. Whether a binding domain specifically reacts with or binds to a target can be easily tested, inter alia, by comparing the reactivity of the binding domain to a target protein or antigen with the reactivity of the binding domain to proteins or antigens other than MUC17 or CD3. Preferably, the binding domains of the present invention essentially or substantially do 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). Superior affinity characteristics compared to other HLE formats are an expected feature of the antibody constructs of the present invention. Such superior affinity consequently suggests an extended in vivo half-life. The longer half-life of the antibody constructs of the present invention may reduce the duration and frequency of administration, which usually contributes to improved patient compliance. This is particularly important because the antibody constructs of the present invention are particularly beneficial for highly debilitated or even multi-disease cancer patients.

[0104] The terms "does not essentially / substantially bind" or "cannot bind" mean that the binding domains of the invention do not bind to proteins or antigens other than MUC17 or CD3, i.e., do not exhibit more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6% or 5% reactivity with proteins or antigens other than MUC17 or CD3, when binding to MUC17 or CD3, respectively, is taken as 100%.

[0105] Specific binding is believed to be mediated by specific motifs within the amino acid sequences of the binding domain and the antigen. Thus, binding occurs as a result of their primary, secondary, and / or tertiary structures, as well as secondary modifications of said structures. The specific interaction of the antigen-interaction site with its specific antigen can result in simple binding of said site to the antigen. Furthermore, the specific interaction of the antigen-interaction site with its specific antigen can 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 responsible for 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 distributed uniformly throughout the variable domains of antibodies, 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) portions of the variable domains are called "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs in three-dimensional space that form 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, connected by three hypervariable regions that form loops connecting and, in some cases, forming 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 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 specific interactions between the antibody and the antigen and thus contribute to the functional activity of the antibody molecule; i.e., they are the primary determinants of antigen specificity.

[0109] The precise definition of CDR boundaries and lengths follows various classification 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 differ, each of these systems has some overlap in the portions that constitute the so-called "hypervariable regions" within the variable sequences. Thus, CDR definitions according to these systems may differ in length and in the boundaries relative to the adjacent framework regions. See, e.g., Kabat (an approach based on sequence variability between species), 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). Another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software.See, for example, Protein Sequence and Structure 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] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main-chain conformation adopted by the antigen-binding (CDR) loop. Comparative structural studies have 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 degree of amino acid sequence variability observed in the majority 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 the 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). Thus, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0111] The term "canonical structure" can also include considerations 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 amino acid residues in antibody variable domains in a consistent manner and, as noted elsewhere herein, is the preferred scheme for application in the present invention. 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 can be described by the Chothia et al. numbering system and / or revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence can be classified into a canonical class that allows, among other things, the identification of an appropriate chassis sequence (e.g., based on the desire to include various canonical structures in a library). The Kabat numbering system for antibody amino acid sequences and the structural considerations described in Chothia et al. (supra) and their significance for interpreting canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a general overview 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 the antigen and the antibody. Using an in vitro selection scheme that changes only the CDR3, the binding properties of the antibody can be changed or which residues contribute to antigen binding can be determined. Therefore, the 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 classical full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The CH domain closest to the 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 and cell-mediated cytotoxicity and complement activation. The Fc region of an antibody is contained within the heavy chain constant domain and can interact, for example, with Fc receptors located on the cell surface.

[0114] The sequences of antibody genes after construction and somatic mutation are highly diverse, and these diverse 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 heavy chains and the V and J segments of light chains. Alternatively, the sequence may be generated from cells in response to, for example, in vitro stimuli that cause rearrangement. Alternatively, some or all of the sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods (see, e.g., U.S. Pat. No. 5,565,332). A repertoire may include only one sequence or may include multiple sequences, including those within a genetically diverse collection.

[0115] The term "Fc portion" or "Fc monomer" in the context of the present invention refers to a polypeptide comprising at least one domain having the function of a CH2 domain and at least one domain having 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 retaining a functional portion of at least one CH2 domain and one CH3 domain, with the CH2 domain located amino-terminal to the CH3 domain. In a preferred embodiment of this definition, an Fc monomer may be a polypeptide constant region comprising a portion of an Ig-Fc hinge region, a CH2 region, and a CH3 region, with the hinge region located amino-terminal to the CH2 domain. The hinge region of the present invention is expected to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by papain digestion of an immunoglobulin region (which, of course, produces a dimer of two Fc polypeptides). In another aspect of this definition, an Fc monomer can be a polypeptide region comprising a portion of a CH2 region and 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 IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgMFc region, an IgAFc region, an IgDFc region, and an IgEFc region (see, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because there is some variation among 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 noted, 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.In the case of 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 can be defined, for example, as including residues D231 (of the hinge domain—corresponding to D234 in Table 1 below) to P476, L476 (for IgG4) at the carboxyl terminus of the CH3 domain, respectively, according to Kabat numbering. Two Fc portions 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 present 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 can be identified by similarity using the Kabat numbering set forth in Table 1. Consistent with the above, it is envisaged that the minimum requirement for hinge domains / regions of the present invention comprises amino acid residues corresponding to the stretch of IgG1 sequence from D231 D234 to P243 according to the Kabat numbering. It is also envisaged that hinge domains / regions of the present invention comprise or consist of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 477) (corresponding to the stretch of 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 eliminated by an N314X substitution, where X is any amino acid other than Q. The substitution is preferably an 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 the 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 described above: N314X, preferably N314G, and / or the additional substitutions V321C and R309C. In a preferred embodiment of the antibody construct of the invention 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 further embodiments 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 have the following sequence EPKSCDKTHTCPPCP (shown in Table 1 and in SEQ ID NO: 487). Accordingly, these core hinge regions are also envisaged in the context of the present invention.

[0122] The locations and sequences of the 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 present invention, the amino acid residues highlighted in bold within 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 monomer") 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 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). It is also preferred that the linker comprises up to 40 amino acid residues, more preferably up to 35 amino acid residues, and 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 a first domain with a second domain, or to fuse a first or second domain with a third domain, the linker is preferably of sufficient length and sequence to ensure that the first and second domains retain their distinct binding specificities independently of each other. For peptide linkers connecting at least two binding domains (or two variable domains) in the antibody construct of the present invention, these linkers preferably contain only a few amino acid residues, e.g., 12 or fewer amino acid residues. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Contemplated peptide linkers with fewer 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 fusing the first and second domains is shown in SEQ ID NO: 1. A preferred embodiment of a peptide linker for fusing the second and third domains is a (Gly)4-linker, also referred to as a 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 present 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 greater (e.g., 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1-12. Characteristics of such 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). Furthermore, peptide linkers that do not promote any secondary structures are preferred. The interconnection of the above domains can be provided, for example, by genetic engineering as described in the Examples. Methods for preparing fused, operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0127] In a preferred embodiment of the antibody construct or 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 present invention are "bispecific single-chain antibody constructs," more preferably bispecific "single-chain Fvs" (scFvs). Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker, as described herein above, which allows them to be produced, using recombinant methods, as a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, e.g., 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 intact or full-length antibodies. Thus, a single-chain variable fragment (scFv) is a fusion protein of the variable region of an immunoglobulin heavy chain (VH) and the variable region of a light chain (VL), typically connected by a short linker peptide of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0129] Bispecific single-chain antibody constructs are known in the art and are 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, and 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 having the format (scFv)2) can be created by linking two scFv molecules (e.g., using a linker as described hereinabove). If these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably referred to as bivalent (i.e., it has two valencies for the same target epitope). If these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably referred to as 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 generate 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, resulting in dimerization of the scFv, 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] Consistent 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. Single domain antibodies comprise 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 developed from heavy chain antibodies found in camels; these comprised V H The cartilaginous fish also have heavy chain antibodies (IgNAR), which are then called V fragments. 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 a human or rodent, 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 to 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 is a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies independently selected from the group comprising: (a) an "scFv-single domain mAb" and (b) an "scFv-single domain mAb"; and (c) an "scFv-single domain mAb" and an "scFv-single domain mAb"; the linker is preferably in the form of a peptide linker. Similarly, an "scFv-single domain mAb" is a monoclonal antibody construct consisting of at least one single domain antibody as defined above and one scFv molecule as defined above. Again, the linker is preferably in the form of a peptide linker.

[0133] The binding of an antibody construct to another given antibody construct in competition 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. Similar to an avidin-coated ELISA plate, each of these beads can be used as a substrate when reacted with biotinylated proteins, on which the assay can be performed. The antigen is coated onto the beads, and then pre-coated with a primary antibody. A secondary antibody is added to confirm any further binding. Possible reading methods 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 recognizing antigens bound to major histocompatibility complex (MHC) molecules and is composed of two distinct 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 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 closely 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 an immunoreceptor tyrosine-based activation motif, or ITAM, which is essential for the signaling ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on chromosome 11 in humans. The most preferred CD3 epsilon epitope is contained within amino acid residues 1 to 27 of the human CD3 epsilon extracellular domain. The antibody constructs according to the present invention are typically and advantageously expected to exhibit only minimal unspecific T cell activation, which is undesirable in specific immunotherapy, which translates into a low risk of side effects.

[0136] Lysis of redirected target cells via recruitment of T cells by multispecific, or at least bispecific, antibody constructs involves the formation of a cytolytic synapse and the delivery of perforin and granzymes. Engaged 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 present 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 MUC17). The target cells can be a cell line (e.g., CHO) that has been stably or transiently transfected with MUC17, e.g., human or macaque MUC17. Typically, EC 50 Values ​​are expected to be lower in target cell lines that express 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 Cytotoxicity can be measured in a Cr release assay (approximately 18 hours of incubation) or a FACS-based cytotoxicity assay (approximately 48 hours of incubation). The incubation time (cytotoxic response) of the assay can be varied. 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 constructs of the present invention is preferably measured in a cell-based cytotoxicity assay. 51 Cytotoxic activity can also be measured by EC 50 The EC values ​​are expressed as half-maximal effective concentrations (the concentration of an antibody construct that induces a cytotoxic response halfway between baseline and maximum). Preferably, the EC values ​​are 0.01 to 0.15 for the MUC17xCD3 bispecific antibody construct. 50 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 for stimulated / enriched CD8 + When T cells are used as effector cells, ECs are significantly higher than unstimulated PBMCs. 50 Those skilled in the art will recognize that values ​​can be expected to be lower. 50 Values ​​can be expected to be lower if target cells express a large amount of MUC17 compared to rats with low target expression. 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 is 50The 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. When human PBMCs are used as effector cells, the EC 50 The value is preferably ≦5000 pM or ≦4000 pM (particularly when the target cells are MUC17-positive human cell lines), more preferably ≦2000 pM (particularly when the target cells are MUC17-transfected cells such as CHO cells), 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 CHO cells 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 present 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," "does not essentially induce lysis," "does not mediate lysis," or "does not essentially mediate lysis" mean that, when the lysis of a MUC17-positive human cell line is taken as 100%, the antibody construct of the present invention does not induce or mediate lysis of more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5% of MUC17-negative cells. This is usually the case at antibody construct concentrations up to 500 nM. Those skilled in the art know how to measure cytolysis without further effort. Furthermore, specific instructions for measuring cytolysis 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 EC values ​​and dimeric forms 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 to human MUC17 and further binds to macaque MUC17, such as cynomolgus monkey (Macaca fascicularis) MUC17, and more preferably to macaque MUC17 expressed on the surface of cells, such as CHO or 293 cells. 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. Affinity can be measured, for example, in a BIAcore assay or a Scatchard assay. Other methods for determining affinity will be familiar to those skilled in the art. The affinity of the first domain for 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] (as determined, for example, 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 present invention binds to human CD3 epsilon and / or macaque CD3 epsilon. In a preferred embodiment, the second domain further binds to marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus Oedipus) or squirrel monkey (Saimiri sciureus) CD3 epsilon. Both marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus) are New World primates belonging to the marmoset (Callitrichidae) family, while squirrel monkeys (Saimiri sciureus) are New World primates belonging to the capuchin (Cebidae) family.

[0146] With respect to the antibody construct of the invention, the second binding domain that 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. Preferably, 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] For the antibody constructs of the 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 as set out in SEQ ID NO: 13 according to the invention.

[0150] It is also preferred that the second domain that binds to CD3 comprises a VH region selected from the group consisting of those set forth in SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008 / 119567 or 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) a VL region as set forth in SEQ ID NO: 35 or 39 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 33 or 37 of WO 2008 / 119567; (c) the VL region set forth in SEQ ID NO: 53 or 57 of WO 2008 / 119567 and the VH region set forth in SEQ ID NO: 51 or 55 of WO 2008 / 119567; (d) a VL region as set forth in SEQ ID NO: 71 or 75 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 69 or 73 of WO 2008 / 119567; (e) a VL region set forth in SEQ ID NO: 89 or 93 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 87 or 91 of WO 2008 / 119567; (f) a VL region as set forth in SEQ ID NO: 107 or 111 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 105 or 109 of WO 2008 / 119567; (g) a VL region set forth in SEQ ID NO: 125 or 129 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 123 or 127 of WO 2008 / 119567; (h) the VL region set forth in SEQ ID NO: 143 or 147 of WO 2008 / 119567 and the VH region set forth in SEQ ID NO: 141 or 145 of WO 2008 / 119567; (i) the VL region set forth in SEQ ID NO: 161 or 165 of WO 2008 / 119567 and the VH region set forth in SEQ ID NO: 159 or 163 of WO 2008 / 119567; and (j) a VL region shown in SEQ ID NO: 179 or 183 of WO 2008 / 119567 and a 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 the antibody construct of the present invention is a CD3-binding second domain comprising the VL region shown in SEQ ID NO:13 and the VH region shown in SEQ ID NO:14.

[0153] According to a preferred embodiment of the antibody construct of the present invention, the first and / or second domain has the following format: a pair of VH and VL domains in the format of a single-chain antibody (scFv). The VH and VL domains are arranged in the order of 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] A preferred embodiment of the above-mentioned antibody construct of the present invention is characterized by a CD3-binding second domain comprising 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 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: 158, and CDR-L3 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, 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: 433, 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: 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 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 envisioned that the antibody comprises a VH region and a VL region selected from the group consisting of:

[0157] It is further envisaged that the first binding domain of the antibody construct of the invention comprises an amino acid sequence selected from the group consisting of those set forth in 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 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, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 412, 413, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 4

[0023] 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 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-chloromercuribenzoate, 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; the reaction is preferably performed 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; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0162] Arginyl residues are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with lysine and arginine epsilon-amino groups.

[0163] The specific modification of tyrosyl residues may be undertaken, particularly for the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane, most commonly using N-acetylimidizole and tetranitromethane 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 the ester with 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield 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. Both forms of these residues are within the scope of this 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 both on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below) and 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. N-linked 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 attachment 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 can be conveniently added to an antibody construct by modifying the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). Modifications can also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). Briefly, it is preferred to modify the amino acid sequence of an antibody construct 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 acids.

[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 with glycosylation capabilities for N- and O-linked glycosylation. Depending on the linkage mode used, sugars can be added to (a) arginine and histidine, (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 can be achieved by the use of various endo- and exo-glycosidases as described by Thotakura 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 prevents the formation of protein-N-glycosidic bonds.

[0173] Other modifications of antibody constructs are also contemplated herein. For example, another type of covalent modification of an antibody construct includes linking the antibody construct to various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, in the manner described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337. In addition, as 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 PEG.

[0174] In some embodiments, covalent modification of the antibody construct of the present invention comprises the addition of one or more labels. To reduce potential steric hindrance, labeling groups can be attached to the antibody construct via spacer arms of various lengths. Various methods for labeling proteins are known in the art and can be used in practicing the present invention. The term "label" or "labeling group" refers to any detectable label. Generally, 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: 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 Isotopic labels, which can be radioactive isotopes or heavy isotopes, such as I) 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 (e.g., 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 GFPs from Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), green fluorescent proteins including EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent proteins (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., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1996, Curr. Biol. 6:178-182), and the like. al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. 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 contain additional domains, e.g., useful for isolating the molecule or relevant for tailoring the pharmacokinetic profile of the molecule. Domains useful for isolating the antibody construct may be selected from peptide motifs or secondarily introduced moieties that can be captured by isolation methods, e.g., isolation columns. 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., Strep II tags), and His tags. All of the antibody constructs disclosed herein may contain 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 can be located, for example, at either the N- or 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. Additionally, PLGA-PEG-PLGA conjugate systems may be combined with polyhistidine tags for sustained release applications and improved pharmacokinetic profiles.

[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 desirable. 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, e.g., 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 if desired. 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 may be 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 location 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 construct 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 construct of the invention. Insertional variants of the antibody construct of the invention include the fusion of an enzyme or polypeptide to the N- or C-terminus of the antibody construct.

[0182] The most important sites for substitutional mutagenesis include, but are not limited to, the CDRs of the heavy and / or light chains, particularly the hypervariable regions, although modifications of the FRs in the heavy and / or light chains are also contemplated. 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 contains 6 amino acids, it is contemplated that 1, 2, or 3 of these amino acids may be substituted. Similarly, if the CDR sequence contains 15 amino acids, it is contemplated that 1, 2, 3, 4, 5, or 6 of these amino acids may be 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 group of residues within the antibody construct (e.g., charged residues such as arg, asp, his, lys, and glu) that affect the interaction of the amino acid with the epitope is identified and 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 demonstrate functional sensitivity to the substitution. Thus, the site or region for introducing an amino acid sequence variant 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 region, and the expressed antibody construct variants are screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites within DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of variants 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 chain, it is preferred that the resulting "substituted" sequence be at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical to the "original" CDR sequence. This means that the degree of identity to the "substituted" sequence depends on the length of the CDR. For example, a CDR having five amino acids is preferably 80% identical to its substituted sequence, since it has at least one substituted amino acid. Thus, the CDRs of an antibody construct may have different degrees of identity to their substituted sequences, e.g., 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 envisioned, as long as the antibody construct retains the ability to bind to MUC17 via the first binding domain and to 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 particularly 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 relation to 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 present 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 side chain bulk. 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 involve exchanging a member of one of these classes for another. Substitution of any cysteine ​​residue not involved in maintaining the proper conformation of the antibody construct, generally with serine, may improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, adding cysteine ​​bond(s) to an antibody may improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0191] For amino acid sequences, sequence identity and / or similarity are determined by standard techniques known in the art, including, but not limited to, the partial sequence identity algorithm of 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 search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), the BestFit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by visual inspection. Preferably, 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 progressive pairwise alignments. 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. This 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 fraction = 0.125, and word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values ​​that are set by the program itself depending on the composition of the individual sequence and the composition of the particular database in which the sequence of interest is being searched, but the values ​​can 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 scores corresponding to approximately 22 bits.

[0195] Generally, the amino acid homology, similarity, or identity between individual variant CDR or VH / VL sequences is at least 60% relative to the sequences set forth herein, and more typically, it is preferred that the homology or identity be increased to at least 65% or 70%, more preferably at least 75% or 80%, and even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even closer to 100%. Similarly, "percent nucleic acid sequence identity" with respect to nucleic acid sequences of binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with nucleotide residues in the coding sequence of the antibody construct. A specific method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with overlap coverage 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 forth herein will be at least 60%, more typically 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 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 percent identity of the antibody construct of the present invention to human germline genes 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 an important feature for reducing the risk of therapeutic proteins eliciting an immune response to the drug in patients undergoing treatment. Hwang & Foote ("Immunogenicity of Engineered Antibodies"; Methods 36 (2005) 3-10) demonstrated that reducing the nonhuman portion of a drug-antibody construct reduces the risk of eliciting anti-drug antibodies in patients undergoing treatment. By comparing a large number of clinically evaluated antibody drugs and corresponding immunogenicity data, humanized antibody V regions have shown a tendency for proteins to be less immunogenic (5.1% of patients on average) than antibodies possessing unmodified nonhuman V regions (23.59% of patients on average). Therefore, for protein therapeutics in the form of V region-based antibody constructs, high identity to human sequences is desirable. To determine this germline identity, Vector NTI software can be used to align the VL V region with the amino acid sequences of human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ), 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 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 research scale conditions, for example in a standard two-step purification process. Preferably, the monomer yield of the antibody constructs 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. Productivities of monomeric and dimeric antibody constructs and calculated monomer percentages can be obtained, for example, by SEC purification steps of culture supernatants derived 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 construct 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 collected from healthy donors using an EDTA-coated syringe. Cellular components are removed by centrifugation, and the upper plasma phase is collected and then pooled. As a control, the antibody construct is diluted in RPMI-1640 medium immediately before the cytotoxicity assay. 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 in an incubator at a concentration of, for example, 100 μg / ml or 250 μg / ml at 37° C. for 7 days. Under these conditions, the antibody construct of the present invention preferably exhibits a dimer fraction 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%, ≦0.5%, or even 0%.

[0202] It is also preferred that the bispecific antibody constructs of the present invention exhibit very low dimer conversion rates after several freeze / thaw cycles. For example, the monomers of the antibody constructs are adjusted to a concentration of 250 μg / ml, for example, in a general-purpose 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 high-speed 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 ≦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%, for example, 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. The thermal stability parameter in terms of the aggregation temperature of an antibody can be determined as follows: An antibody solution at 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, with the radius measurement being continuously acquired. The increase in radius, which indicates 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 antibody constructs. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC instrument. The energy uptake of samples containing the antibody constructs is recorded from 20°C to 90°C and compared to samples containing formulation buffer only. The antibody constructs are adjusted to a final concentration of 250 μg / ml, for example, 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 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 2.5 mg / 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 to 6.0. The higher the tolerance of the antibody construct at non-physiological pH, e.g., about pH 6.0, the higher the recovery rate of the antibody construct eluted from the ion exchange column relative to the total amount of loaded protein. The recovery rate 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 will 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 average tumor volume is approximately 100 mm 3 When the total number of CD3+ T cells reaches 10, approximately 2 × 10 cells are injected into the peritoneal cavity of the animals. 7 Mice are implanted with T cells by injection of 1000 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 (which receive effector cells) to monitor the effect of T cells alone on tumor growth. Mice with a mean tumor volume of approximately 200 mm 3 Antibody treatment begins when tumor size reaches 100%. The mean tumor size of each treatment group on the treatment initiation date should not be statistically different from any other group (analysis of variance). Mice are treated with the MUC17xCD3 bispecific antibody construct at 0.5 mg / kg / day via intravenous bolus injection for approximately 15-20 days. Tumors are measured by caliper during the study, and progression is assessed by intergroup comparison of tumor volume (TV). Tumor growth inhibition (T / C) [%] is determined by calculating TV as T / C% = 100 × (median TV of the analyzed group) / (median TV of the control group 2).

[0209] Those skilled in the art know how to vary or adapt certain parameters of this test, such as the number of tumor cells injected, the injection site, the number of implanted human T cells, 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, 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 present 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 having the general structure RSSR. This linkage, also known as an S-S bond or disulfide bridge, is obtained by coupling of two thiol groups of cysteine ​​residues. With regard to the antibody construct of the present invention, it is particularly preferred that the cysteines that form 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 present invention, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of the glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid other than Q. The substitution is preferably N314G. In a more preferred embodiment, the 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 present 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 respect to the constructs of the present invention, it is preferred that the CH2 domain within the third domain of the antibody construct of the present invention comprises an intradomain cysteine ​​disulfide bridge at Kabat positions 309 and 321 and / or the glycosylation site at Kabat position 314 is removed, preferably by an N314G substitution.

[0215] In a further preferred embodiment of the invention, 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 the glycosylation site at Kabat position 314 is eliminated by an N314G substitution. Most preferably, the polypeptide monomer of the third domain of the antibody construct of the invention 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 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 nanobodies or single variable domain antibodies comprising only one variable domain, which may be, for example, 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 greater (e.g., 2 or 3). A particularly preferred linker for fusing the first and second domains to the third domain is shown in SEQ ID NO: 1.

[0219] In a preferred embodiment, the antibody constructs of the invention comprise, in amino to carboxyl order: (a) First domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) 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) the 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 present invention comprise a first domain that binds to MUC17, preferably 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, a first domain according to the present invention preferably binds to MUC17 when expressed by a cell or cell line that naturally expresses MUC17 and / or by a cell or cell line that has been transformed or transfected (stably or transiently) with MUC17. 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 ECD. "MUC17 extracellular domain" or "MUC17 ECD" refers to a region or sequence of MUC17 that is essentially free of the transmembrane and cytoplasmic domains of MUC17. Those skilled in the art will understand 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 connection with 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, and 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 the third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17 to 24; Includes.

[0224] Consistent with this preferred embodiment, the first and second domains fused to the single-chain polypeptide via a peptide linker comprise a sequence 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 aspect, the antibody construct of the invention comprises an antibody selected from the group consisting of SEQ ID NOs: 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 biological polymer composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (e.g., cDNA) and RNA (e.g., 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 or single-stranded, linear or circular. It is preferably contained in a vector contained within a host cell. The host cell is capable of expressing the antibody construct, for example, after transformation or transfection with the vector or polynucleotide of the present invention. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a regulatory sequence.

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

[0228] The present invention further provides vectors comprising the polynucleotides / nucleic acid molecules of the present invention. A vector is a nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into cells. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Genetically engineered vectors generally contain an origin of replication, a multiple cloning site, and a selection marker. The vector itself is generally a nucleotide sequence, typically 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, and protein purification tags. Vectors, called expression vectors (expression constructs), are specifically designed for the expression of transgenes in target cells and generally contain 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 that are 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 pre-protein 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 promote 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. Linking 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 intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term is most often used for non-viral methods in eukaryotic cells. Transduction is often used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves creating transient pores, or "holes," in the cell membrane to allow uptake of the substance. Transfection can be achieved using calcium phosphate, by electroporation, by cell compression, or by mixing cationic lipids with the substance to form liposomes, which fuse with the cell membrane and accumulate the cargo inside.

[0232] The term "transformation" is used to refer to 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 of exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane and subsequent incorporation. 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] The present invention further 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, exogenous nucleic acid molecules, and polynucleotides encoding the antibody construct of the present invention; and / or the antibody construct itself. Introduction of the respective substance into the cell is accomplished by transformation, transfection, etc. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Because certain modifications may occur in successive generations due to either spontaneous, accidental, or deliberate mutation, 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 be 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 constructs of the present invention can be produced in bacteria. After expression, the antibody constructs 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 carried out, 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), and others. Kluyveromyces hosts such as Kluyveromyces spp. (European Patent No. 36906), K. thermotolerans, and K. marxianus; Yarrowia spp. (European Patent No. 402226); Pichia pastoris (European Patent No. 183070); Candida spp.; Trichoderma reesia (European Patent No. 244234); Neurospora crassa; Schwanniomyces occidentalis; Schwanniomyces such as A. occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0236] Suitable host cells for expression of the glycosylated antibody constructs of the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant 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 (silkworm). Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as the 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 producing proteins in plant cell cultures are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Owen 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 32:979-986.

[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 are the 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 that allow 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 producing 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, antibody constructs can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the antibody construct is produced intracellularly, the first step is to remove particulate debris from 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 Escherichia coli (E. coli). Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, supernatants from such expression systems are 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 contaminants.

[0242] The antibody constructs of the present 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 recovered, other protein purification techniques, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available. 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] The present invention further provides a pharmaceutical composition comprising an antibody construct of the present invention or an antibody construct produced by a 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 (pharmaceutically 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 contain a pharmaceutically acceptable carrier. Generally, as used herein, "pharmaceutically acceptable carrier" refers to any aqueous or 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, liposomes, dispersion media and coatings 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 compositions 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 one or more additional excipients, such as those illustratively 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 to adjust the physical, chemical, or biological properties of the formulation, such as adjusting viscosity, and / or to improve efficacy and / or stabilize such formulations, as well as against degradation and damage due to stresses encountered during and after manufacture, transportation, storage, preparation prior to use, and administration.

[0248] In certain embodiments, pharmaceutical compositions may contain formulating materials intended to modify, sustain, or protect, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulating materials may include, but are not limited to, the following: charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and / or histidine, such as 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, such as 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); ·Isotonic 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, or 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 cosolvents (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, tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be detergents, preferably with a molecular weight of >1.2 KD, and / or polyethers, preferably with a molecular weight of >3 KD; 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, (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 an antibody construct, optionally wherein the third domain comprises two polypeptide monomers, each comprising a hinge, a CH2 domain, and a CH3 domain, and wherein the two polypeptide monomers are fused to each other via a peptide linker; (b) at least one buffering agent; (c) at least one sugar; 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 contemplated in the context of the present invention that the at least one buffering agent is present at a concentration ranging from 5 to 200 mM, more preferably from 10 to 50 mM. It is also contemplated 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 contemplated in the context of the present invention that the disaccharide is selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, and combinations thereof. It is further contemplated in the context of the present invention that the sugar alcohol is sorbitol. It is also contemplated in the context of the present invention that the at least one sugar is present at a concentration ranging from 1 to 15% (m / V), preferably from 9 to 12% (m / V).

[0251] It is also contemplated in 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 contemplated in the present invention that the at least one surfactant is present at a concentration ranging from 0.004 to 0.5% (m / V), preferably ranging from 0.001 to 0.01% (m / V). It is also contemplated in the present invention that the pH of the composition is ranging from 4.0 to 5.0, preferably 4.2. It is also contemplated in the present invention that the pharmaceutical composition has an osmolality ranging from 150 to 500 mOsm. It is further contemplated in 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 to 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 to 8 mg / ml, preferably 0.2 to 2.5 mg / ml, more preferably 0.25 to 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, for example, amino acids may act as buffers, stabilizers and / or antioxidants; mannitol may act as a bulking agent and / or tonicity enhancer; sodium chloride may act as a delivery vehicle and / or tonicity enhancer, etc.

[0255] It is envisioned that the compositions of the present invention may contain, in addition to the polypeptides of the present invention as defined herein, further biologically active agents depending on the intended use of the composition. Such agents may be drugs known in the art that act on the gastrointestinal system, drugs that act as cytostatics, drugs that prevent hyperuricemia, drugs that inhibit immune responses (e.g., corticosteroids), drugs that modulate inflammatory responses, drugs that act on the circulatory system and / or cytokines. It is also envisioned that the antibody constructs of the present invention may be 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 on, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence 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 a 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 ingredients 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 present composition 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). Additionally, 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 intended, therapeutic compositions 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 pharmaceutically 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, appropriately preserved. In certain embodiments, the formulation may include a formulation of the desired molecule with an agent capable of providing controlled or sustained release of the product, which can be delivered via depot injection, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid, which has the effect of enhancing duration in the circulation, may also be used. In certain embodiments, the desired antibody construct may be introduced using an implantable drug delivery device.

[0258] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody constructs of the invention in sustained- or controlled-delivery / release formulations. Techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible 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 comprise semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices can include polyesters, hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919 and 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). Sustained-release compositions can also include liposomes, which can 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 a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0261] Another aspect of the present 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 are available for protein stabilization and formulation materials and methods useful in this regard, 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 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 sections regarding the same excipients and processes as in the self-buffering protein formulations according to the present invention.

[0262] Salts may be used in certain embodiments of the present invention, for example, to adjust the ionic strength and / or tonicity of a formulation and / or to improve the solubility and / or physical stability of proteins or other components of a composition according to the present invention. As is well known, ions can stabilize proteins in their 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 their denatured state, particularly by binding to the protein's denatured peptide bond (--CONH). Furthermore, ionic interactions with charged and polar groups in proteins can 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 developed and can be used in formulating pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropics." Destabilizing solutes are called "chaotropics." Kosmotropes are commonly used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate ("salting out") proteins from solution. Chaotropes are commonly used to denature and / or solubilize ("salting in") proteins. The relative effects of an ion on "salting in" and "salting out" define its position on the Hofmeister series.

[0264] Free amino acids can be used in the antibody constructs of the formulations of the present invention according to various embodiments as bulking agents, stabilizers, antioxidants, and 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 during 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, as well as polyhydric alcohols such as glycerol and propylene glycol, and for purposes of this discussion, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. They are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful for adjusting the tonicity of formulations. Among the polyols useful in selected embodiments of the present invention is mannitol, which is commonly used in lyophilized formulations to ensure cake structural stability. Mannitol ensures cake structural stability. It is generally used in conjunction with a lyoprotectant, such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting tonicity and stabilizers for protection against freeze-thaw stress during transportation or bulk preparation in manufacturing processes. 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, as they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a cryoprotectant, and in this regard can be used in the present invention.

[0266] Embodiments of the antibody construct of the present invention further comprise a surfactant. Protein molecules can be prone to surface adsorption and denaturation and subsequent 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 traditionally used to prevent, minimize, or reduce surface adsorption. Surfactants useful 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, as any given surfactant will typically stabilize some proteins and destabilize others.

[0267] Polysorbates are prone to oxidative degradation and often contain sufficient peroxides as supplied to cause oxidation of protein residue side chains, particularly methionine. Therefore, polysorbates should 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] Embodiments of the antibody construct of the formulation of the present invention further comprise one or more antioxidants. Detrimental oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and by avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Particularly useful antioxidants in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations of the present invention are preferably water-soluble and maintain activity throughout 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 disrupt intramolecular disulfide bonds, among other things. Therefore, antioxidants for use in the present invention are selected, inter alia, to eliminate or sufficiently reduce the possibility of damaging proteins in the formulation.

[0269] The formulations of the present invention may contain metal ions, which are protein cofactors and are required to form protein coordination complexes, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some protein degradation processes. However, metal ions also catalyze the physical and chemical processes that degrade 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 due to the presence of Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 The aggregation can be destabilized by Al+3 It can be increased by ions.

[0270] Embodiments of the antibody construct formulations of the present invention further comprise one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations involving multiple withdrawals from the same container. Their primary function is to inhibit microbial growth and ensure product sterility over the shelf life or usage period of the formulation. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While preservatives have a long history of use with small molecule parenteral drugs, developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single-use only. However, the possibility of multi-dose formulations offers the added benefits of patient convenience and increased marketability. Human growth hormone (hGH) is a good example, where the development of a preservative-treated formulation led to the commercialization of a more convenient multi-use injection pen. 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 must 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 confer antimicrobial efficacy without compromising protein stability.

[0271] As expected, developing a liquid formulation containing a preservative is more challenging than a lyophilized formulation. 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, significantly minimizing the associated stability risks. For liquid formulations, the preservative's effectiveness and stability should be maintained throughout the product's shelf life (approximately 18-24 months). It is important to note that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0272] The antibody constructs disclosed herein can 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 liposomes 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, e.g., 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 reverse-phase evaporation using 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 present invention can be conjugated to liposomes via a disulfide exchange reaction as described by Martin et al. J. Biol. Chem. 257:286-288 (1982). Optionally, a chemotherapeutic agent is contained within the liposome. 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 (e.g., lyophilized) that is reconstituted prior to administration.

[0274] The biological activity of the pharmaceutical compositions defined herein can 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 compositions of the present invention, for example, using standardized NCI response criteria. The success of therapy using the pharmaceutical compositions of the present invention, or in vivo efficacy, refers to the effectiveness of the composition for its intended purpose, i.e., its ability to cause its desired effect, i.e., the depletion of pathological cells, e.g., tumor cells. In vivo efficacy can be monitored by established standard methods for each disease entity, including, but not limited to, white blood cell counts, differential counts, fluorescence-activated cell sorting, and bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used.Additionally, 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 pathological condition. Pharmacokinetic parameters of drugs that affect a drug's ability 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 anticipated 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 "canonical" non-HLE versions of the 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 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 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 between administration of a drug and when the drug can be detected and measured in the 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 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 specified, for example, in the publication by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12).

[0278] In a preferred embodiment of the present 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 present invention relative to the quality of corresponding state-of-the-art antibody constructs can be tested using different systems. These tests are understood to be in accordance with the "ICH Harmonized Tripartite Guideline: Stability Testing of Biotechnological / Biological Products Q5C and Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products Q6B," and are selected to provide a stability-indicating 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 resulting purity value is method-dependent. For stability testing purposes, purity tests should be combined with methods for determining degradation products.

[0279] The quality of a pharmaceutical composition comprising an antibody construct of the present invention can be assessed, for example, by analyzing the content of soluble aggregates in solution (HMWS by size exclusion). Stability for at least 4 weeks at about -20°C 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 formulation components as described below. ·formulation: Potassium phosphate pH 6.0, L-arginine hydrochloride, trehalose dihydrate, polysorbate 80 Other examples of evaluating the stability of antibody constructs of the present invention in the form of pharmaceutical compositions are provided in the accompanying Examples 4-12. In these examples, embodiments of the antibody constructs of the present invention are tested under different stress conditions in different pharmaceutical formulations, and the results are compared with other bispecific T cell-engaging antibody constructs in half-life extension (HLE) formats known in the art. In general, antibody constructs provided in a specific FC format according to the present invention are generally expected to be more stable against a wide range of stress conditions, such as temperature and light stress, compared with both antibody constructs provided with different HLE formats and antibody constructs without any HLE format (e.g., "canonical" antibody constructs). 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 recognize, such improved stability against stresses that are difficult to avoid in clinical practice will result in safer antibody constructs in clinical practice, as fewer degradation products will be produced. Consequently, the aforementioned improved stability also means improved safety.

[0281] One embodiment provides an antibody construct of the invention or made according to a 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 a patient in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of the formulations to the body, isolated tissues, or cells of a patient with 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 defined herein below, by administration of an antibody construct according to the present invention to a subject in need thereof. Such improvement may be seen as a slowing or halting of the 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 defined herein below, in a patient having such a tumor or cancer, 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 the antibody constructs or pharmaceutical compositions 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, although not necessarily, forming a mass. When it forms a mass, it is commonly called a "tumor." A neoplasm or tumor can be benign, potentially malignant (precancerous), or malignant. Malignant neoplasms are commonly 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" includes metastases to 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 a viral infection in a subject. 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 produced according to the methods 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" includes subjects already with the disorder as well as subjects in which the disorder is to be prevented. A subject in need or a "patient" includes human 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, 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, pinna / 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, extraamniotic, intraarticular, 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, e.g., bolus injection, or by infusion, e.g., 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. Patent 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 can be achieved by a miniature pump system worn by the patient to regulate the influx of a therapeutic agent into the patient's body. Pharmaceutical compositions comprising antibody constructs of the present invention can be administered using such pump systems. 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 pump systems, a temporary interruption in the otherwise uninterrupted flow of therapeutic agent into the patient's body may result. Even in such cases, the administration step before cartridge replacement and the administration step after cartridge replacement would still be considered within the meaning of the pharmaceutical means and methods of the present invention, together constituting the "uninterrupted administration" of such therapeutic agent.

[0293] Continuous or uninterrupted administration of the antibody construct of the present invention can be intravenous or subcutaneous administration using a fluid delivery device or miniature pump system, which includes a fluid pumping mechanism for pumping fluid from a reservoir and a drive mechanism for driving the pumping mechanism. A pump system for subcutaneous administration can include a needle or cannula for penetrating the patient's skin and delivering the suitable composition into the patient's body. The pump system can be fixed or attached directly to the patient's skin, whether it be a vein, artery, or blood vessel, allowing for direct contact between the pump system and the patient's skin. This pump system can be attached to the patient's skin for 24 hours to several days. In some cases, the pump system can 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 can be 0.1 to 50 ml.

[0294] Continuous administration can also be transdermal, by means of 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 should be 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 adjacent to the first used patch immediately before removing the first used patch, and the replacement of the first used patch can be completed at the same time.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 an appropriate liquid prior to administration, for example, bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was present before lyophilization.

[0296] The compositions 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 in which the antibody constructs of the present invention exhibiting cross-species specificity described herein are administered to non-chimpanzee primates, such as macaques, in increasing doses. As described above, the antibody constructs of the present invention exhibiting cross-species specificity described herein have the advantage that they can be used in the same form in preclinical studies in non-chimpanzee primates and as drugs in humans. The dosing regimen will be determined by the attending physician based on clinical factors. As is well known in the medical field, the dosage administered to a given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly.

[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 dosage 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 status of the patient's own immune system. The appropriate dosage can be administered to the patient in a single administration or multiple administrations, 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 an antibody construct of the invention preferably reduces the severity of disease symptoms, increases the frequency or duration of symptom-free periods, or prevents impairment or disability due to disease affliction. With respect to treating diseases associated with MUC17 expression as described herein above, a therapeutically effective amount of an 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, e.g., other proteinaceous and non-proteinaceous drugs, which may be administered simultaneously with the composition comprising the antibody construct of the present invention as defined herein, or may be administered separately at predetermined time intervals and dosages before or after administration of said antibody construct.

[0301] As used herein, the term "effective and non-toxic dose" refers to a tolerated dose of an antibody construct of the present invention that is sufficient to result in depletion of pathological cells, tumor elimination, tumor regression, or disease stabilization without or essentially without significant toxic effects. Such an effective and non-toxic dose can be determined, for example, by dose escalation studies described in the art, and should be below the dose that induces serious adverse side effects (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 effects 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 the 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 assessed, for example, periodically during treatment and follow-up. Measurements include clinical evaluations, such as organ findings and screening for laboratory abnormalities. Clinical evaluations can be performed, and deviations from normal findings can be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings can include, for example, criteria for allergy / immunology, blood / bone marrow, cardiac arrhythmia, coagulation, etc., as set forth in the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profile, and urinalysis, as well as tests of other body fluids, such as serum, plasma, lymph, or spinal fluid. Thus, safety can be assessed, for example, by 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 can 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 of July 16, 1997; ICH Harmonized Tripartite Guideline; ICH Steering Committee meeting.

[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 arrangement, one of which corresponds to an antibody construct, pharmaceutical composition, vector, or host cell of the present invention. A kit can therefore be described as a set of products and / or implements sufficient to achieve a particular purpose that can be sold as a single item.

[0307] The kit may comprise one or more containers (e.g., vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing an antibody construct or pharmaceutical composition of the invention in a dosage amount suitable for administration (see above). 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, a reference to a "reagent" includes one or more of such different reagents, and a reference to a "method" includes reference to equivalent steps and methods known to those skilled 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 this invention.

[0311] The term "and / or", wherever used herein, 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, although the term also includes specific numbers, for example, about 20 includes 20.

[0313] The terms "less than" or "greater than" are inclusive of specific numbers. 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 that 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 sometimes the term "having," when used herein.

[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 present invention, which is defined solely by the claims.

[0317] All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), cited throughout the text of this specification, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference contradicts or is 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 invention in any way. The present invention includes, but is not limited to, 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 extracellular epitopes of human and macaque CD3ε chains 1. A bispecific antibody construct comprising: [Aspect 2] 2. The bispecific antibody construct of aspect 1, further comprising a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 domain, and a CH3 domain, wherein said two polypeptide monomers are 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 of embodiment 2 or 3, comprising: [Aspect 5] 5. The bispecific antibody construct of 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 aspects 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 of any one of aspects 1 to 11, wherein the first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 528 (aa 4171-4296 according to Uniprot Q685J3 numbering). [Aspect 13] 13. The bispecific antibody construct of any one of aspects 1 to 12, wherein the first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 529 (aa 4189-4291 according to Uniprot Q685J3 numbering). [Aspect 14] 14. The bispecific antibody construct of any one of aspects 1 to 13, wherein the first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 530 (aa 4131-4243 according to Uniprot Q685J3 numbering). [Aspect 15] 15. The bispecific antibody construct of any one of aspects 1 to 14, wherein the first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 531 (aa 4244-4389 according to Uniprot Q685J3 numbering). [Aspect 16] 16. The bispecific antibody construct of any one of aspects 1 to 15, wherein the first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO: 530 (aa 4131-4243 according to Uniprot Q685J3 numbering) but does not bind to an epitope within MUC17 corresponding to SEQ ID NO: 531 (aa 4244-4389 according to Uniprot Q685J3 numbering). [Aspect 17] 17. The bispecific antibody construct of 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 (aa 4171-4390 according to uniprot Q685J3 numbering) or SEQ ID NO: 533 (aa 4184-4390 according to uniprot Q685J3 numbering), but does not bind to an epitope in MUC17 corresponding to SEQ ID NO: 534 (aa 4291-4390 according to uniprot Q685J3 numbering) or an epitope in MUC17 corresponding to SEQ ID NO: 535 (aa 4341-4390 according to uniprot Q685J3 numbering). [Aspect 18] The ratio between cytotoxicity and binding affinity (EC 50 / K D18. The bispecific antibody construct according to any one of aspects 1 to 17, wherein β-actin (β)*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(R)*1000 is less than 125, said cytotoxicity is determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and said binding affinity is 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(R)*1000 is less than 21, said cytotoxicity is determined in NUGC-4 cells as target cells and huPBMCs as effector cells, and said binding affinity is 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 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, CDR-L2 as set forth in SEQ ID NO: 290, 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...

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, wherein the first domain comprises a VH region comprising 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, and the first domain comprises a VL region comprising 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; 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, wherein the two polypeptide monomers are fused to each other via a peptide linker.

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

4. The third domain comprises, 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, 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; or (ii) the first domain comprises two antibody variable domains, and the second domain comprises one antibody variable domain; A bispecific antibody construct according to any one of claims 1 to 7.

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 of 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. The bispecific antibody construct of claims 2, 4 to 7, and 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. 13. The bispecific antibody construct of any one of claims 1 to 12, wherein said first domain comprises a VL region as set forth in SEQ ID NO: 183 and a VH region as set forth in SEQ ID NO:

182.

14. 14. The bispecific antibody construct of any one of claims 1 to 13, comprising the amino acid sequence of SEQ ID NO:

184.

15. an amino acid sequence consisting of SEQ ID NO: 185 or 186; 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 of claim 1, wherein

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 described in claim 16 or the vector described in 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 that allow expression of the 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 of 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 a bispecific antibody construct according to any one of claims 1 to 15, a polynucleotide according to claim 16, a vector according to claim 17, and / or a host cell according to claim 18.

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