Antibodies against mucin 17 and uses thereof

Abstract

The present invention relates to antibodies that bind to human and monkey mucin 17 (MUC17). Furthermore, the present invention relates to detection systems comprising such antibodies. These antibodies or detection systems can be used for detecting or quantifying MUC17, for diagnosing diseases associated with MUC17, for patient stratification, monitoring disease progression, and evaluating treatment response.

Description

Technical Field

[0001] This invention relates to antibodies that bind to mucin 17 (MUC17). Furthermore, this invention relates to detection systems comprising such antibodies. These antibodies or detection systems can be used to detect or quantify MUC17, for the diagnosis of MUC17-associated diseases, for patient stratification, for monitoring disease progression, and for evaluating treatment response.

[0002] Mucins have been identified as biomarkers of interest for inflammation and cancer. Mucins are high-molecular-weight glycoproteins characterized by high levels of O-glycosylation at serine and threonine residues within their tandem repeat domains (Johansson and Hansson, Nat. Rev. Immunology, 2016). At least 20 members of the mucin family exist, including secretory 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; Hatrup and Gendler, Annu. Rev. Physiol, 2008). Transmembrane mucins also play a role in cell signaling (including the regulation of proliferation and apoptosis) and in tumorigenesis (Hollingsworth and Swanson, Nat. Rev. Cancer [Nature Review: Cancer] 2004). Among mucins, mucin 17 (MUC17) was the first transmembrane mucin identified by its homology with MUC3 (Gum et al., Biochem. Biophys. Res. Comm. [Biochemical and Biophysical Research Communications] 2002).

[0003] Analysis of the complete coding sequence of MUC17 revealed that it possesses a large extracellular domain consisting of a central region of 61 tandem repeats, an epidermal growth factor (EGF) domain, a sea urchin spermin, enterokinase, and agglutinin (SEA) domain, as well as 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 80-amino acid cytoplasmic tail within the cell (Moniaux et al., J. Biol. Chem. 2006). Expression of MUC17 in healthy adults is confined to the apical surface of intestinal epithelial cells lining the intestine or mature absorptive epithelial cells (Moniaux et al., J. Biol. Chem. 2006; Johanasson and Hansson, Nat. Rev. Immunology 2016). MUC17 is also expressed in 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 maintaining the integrity of the mucosal barrier in the intestine, such as through mucosal restoration (Luu et al., Int. J. Biochem. Cell Biol. 2010; Resta-Lenert et al., Am. J. Physiology 2011; Johanasson and Hansson, Nat. Rev. Immunology 2016).

[0004] MUC17 is aberrantly expressed in several cancers. MUC17 mRNA has been shown to be expressed in one pancreatic cancer cell line and three colon cancer cell lines (Gum et al., 2002). Immunohistochemical studies have confirmed the expression of MUC17 protein in pancreatic cancer (Moniaux et al., 2006). However, in colon cancer, MUC17 protein expression has been reduced (Senapati et al., J. Clin. Pathol. [Journal of Clinical Pathology] 2010). Nevertheless, the expression pattern of MUC17 makes it a potential target for the treatment of various forms of malignancy. Clinical trials using anti-MUC17 antibody constructs have been initiated to test its applicability for the treatment of gastrointestinal cancer and gastroesophageal junction cancer (clinicaltrials.gov; NCT04117958). Summary of the Invention

[0005] Given the conflicting implications in the literature regarding MUC17 as a potential target for pathological symptoms, the objective of this invention is to clearly identify specific symptoms associated with MUC17 upregulation and to provide selectively binding conjugates (e.g., antibodies) for use as diagnostic tools in MUC17-related symptoms, preferably for detecting / diagnosing said specific symptoms and / or for monitoring disease status before, during, and after therapeutic inventions. Monitoring is used to determine the success of a given treatment and allows attending physicians to determine whether the treatment is effective or insufficient and whether modifications are needed.

[0006] Ideally, a diagnostic antibody binds to a region that resembles the region recognized or targeted by the therapeutic antibody construct. Because tumor antigens themselves can exhibit different splicing patterns or have mutations affecting the three-dimensional structure of the target, it is desirable to verify the presence of the therapeutic antibody's target in a given patient and whether that patient is eligible for such treatment. When the target region of the tumor antigen is absent in a given patient, treatment with a selectively binding therapeutic antibody construct may not be advantageous given the potential adverse events associated with treatment. Furthermore, the patient will not undergo inappropriate and potentially detrimental, exhausting, and costly treatment, but could instead be treated with more likely effective alternative therapies. It should be remembered that patients receiving immunotherapy are often severely ill. These patients are frequently refractory to first-line therapy or have experienced relapse and / or have metastases that do not respond to initial therapy. It is undesirable to treat such patients without a clear diagnostic basis demonstrating the rationality and medical justification for treatment with immunotherapeutic compounds.

[0007] Therefore, the present invention provides an antibody that binds to MUC17. This antibody can be used in diagnostic methods on tissue samples obtained from individuals, particularly from patients suspected of having neoplastic diseases associated with and / or identifiable by abnormal MUC17 expression, such as gastrointestinal cancer, pancreatic cancer, etc.

[0008] This antibody can also serve as a diagnostic tool for detecting MUC17 in laboratory animals, such as in previously euthanized animals with cancer.

[0009] Furthermore, the present invention provides a polynucleotide encoding the antibody construct, a vector containing the polynucleotide, a host cell expressing the construct, a diagnostic composition containing the construct, and a kit containing the antibody of the present invention as a diagnostic tool and other components, including but not limited to at least one of a second antibody, enzyme, buffer, instruction manual, calibration tool, control, etc.

[0010] Embodiments of the present invention

[0011] In a first embodiment, the present invention relates to an antibody that binds to human MUC17 as depicted in SEQ ID NO:1, wherein the antibody binds to the cell surface-associated MUC17 protein.

[0012] In a second embodiment, the present invention relates to an antibody that binds to human MUC17 as depicted in SEQ ID NO:1, wherein the antibody binds to cell surface-associated MUC17 protein, and wherein the antibody comprises a variable heavy chain containing the CDR3 region depicted in SEQ ID NO:4.

[0013] In a third embodiment, the present invention relates to antibodies according to Examples 1 and 2, wherein the antibodies further comprise heavy chain CDR1 and CDR2 regions as depicted in SEQ ID No: 2 and 3 and / or light chain CDR1, CDR2 and / or CDR3 regions as depicted in SEQ ID No: 6, 7 and 8.

[0014] In a fourth embodiment, the present invention relates to an antibody according to any one of the foregoing embodiments, wherein the monoclonal antibody comprises a VH region and / or a VL region consisting of the sequences depicted in SEQ ID NO:5 and 9.

[0015] In a fifth embodiment, the present invention relates to an antibody according to any one of the foregoing embodiments, wherein the antibody is a monoclonal antibody.

[0016] In a sixth embodiment, the present invention relates to an antibody according to any one of the foregoing embodiments, wherein the antibody specifically binds to human and cynomolgus monkey MUC17 in immunohistochemical assays, cells expressing MUC17 in fluorescence-activated cell sorting assays, and fixed and permeabilized cells expressing MUC17, optionally wherein the antibody does not bind to secreted form huMUC17.

[0017] In a seventh embodiment, the present invention relates to an antibody according to any one of the foregoing embodiments, wherein the antibody comprises the VH region included in SEQ ID NO:5 and the VL region according to SEQ ID NO:9.

[0018] In the eighth embodiment, the present invention relates to an antibody according to any one of the foregoing embodiments, the antibody being an IgG, IgD, IgE, IgM or IgA antibody, preferably an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody.

[0019] Within the embodiments described, and in the context of the present invention, it is further envisioned to provide an antibody construct wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to amino acids 4171 to 4296 numbered according to uniprot Q685J3.

[0020] Within the embodiments described, and within the context of the present invention, it is also contemplated to provide an antibody construct wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to amino acids 4184 to 4291 numbered according to uniprot Q685J3.

[0021] Within the embodiments described, and in the context of the present invention, it is further envisioned to provide an antibody construct wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to amino acids 4131 to 4243 numbered according to uniprot Q685J3.

[0022] Within the embodiments described, and within the context of the present invention, it is also contemplated to provide an antibody construct wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to amino acids 4244 to 4389 numbered according to uniprot Q685J3.

[0023] Within the embodiments described herein, in the context of the present invention, it is further envisioned to provide an antibody construct wherein a first domain of the antibody construct binds to epitopes within MUC17 corresponding to amino acids 4131 to 4243 numbered according to uniprot Q685J3, but does not bind to epitopes within MUC17 corresponding to amino acids 4244 to 4389 numbered according to uniprot Q685J3.

[0024] Within the embodiments described herein, in the context of the invention, it is also contemplated to provide an antibody construct wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to amino acids 4171 to 4390 or 4184 to 4390 according to uniprot Q685J3, but not to an epitope within MUC17 corresponding to amino acids 4291 to 4390 or 4341 to 4390 according to uniprot Q685J3.

[0025] In the ninth embodiment, the present invention relates to a polynucleotide that encodes an antibody according to any one of the foregoing embodiments.

[0026] In the tenth embodiment, the present invention relates to a carrier comprising the polynucleotide according to Example 9.

[0027] In the 11th embodiment, the present invention relates to host cells transformed or transfected with a polynucleotide according to the 10th embodiment or with a vector according to the 9th embodiment.

[0028] In the 12th embodiment, the present invention relates to a process for producing an antibody according to any one of Examples 1 to 8, the process comprising culturing a host cell as described in Example 10 under conditions that allow the expression of the antibody and recovering the resulting antibody from the culture.

[0029] In the 13th embodiment, the present invention relates to a hybridoma that produces an antibody according to any one of the preceding embodiments one to eight.

[0030] In the 14th embodiment, the present invention relates to a composition comprising an antibody produced according to any one of Examples 1 to 8 or according to the process described in Example 12.

[0031] In the 15th embodiment, the present invention relates to a detection system comprising an antibody produced according to any one of Examples 1 to 8 or according to the process described in Example 12.

[0032] In the 16th embodiment, the present invention relates to the use of antibodies produced according to any one of Examples 1-8 or the process described in Example 12, or the detection system described in Example 15, in a diagnostic method.

[0033] In the 17th embodiment, the present invention relates to an antibody produced according to any one of Examples 1-8 or the process described in Example 12, a composition described in Example 14, or a detection system described in Example 15, which is used in a method for detecting tumor growth.

[0034] In the 18th embodiment, the present invention relates to an antibody or detection system for use in a method for detecting tumor growth according to Example 17, the method comprising determining the expression level of MUC17 in samples from a patient suspected of having cancer and negative control samples and optionally positive control samples.

[0035] In the 19th embodiment, the present invention relates to an antibody or detection system for use in a method for detecting tumor growth according to any one of Examples 17 and 18, the method comprising determining the expression level of MUC17 in samples from a patient suspected of having cancer and negative control samples, further comprising comparing the expression level of MUC17 between these samples, optionally wherein the expression level in the negative control and / or positive control may be derived from stored data in at least one negative control sample and / or at least one positive control sample obtained in the method for detecting tumor growth, the method comprising determining the expression level of MUC17, further optionally wherein the expression level in the negative control sample and / or positive control sample may be derived from stored data including the average expression level of more than one negative control sample and / or more than one positive control sample obtained in the method for detecting tumor growth, the method comprising determining the expression level of MUC17.

[0036] In the 20th embodiment, the present invention relates to an antibody or detection system for use in a method for detecting tumor growth according to any one of Examples 17 and 19, the method comprising determining the expression level of MUC17 in a sample, wherein the sample is a solid tissue sample or a liquid tissue sample.

[0037] In the 21st embodiment, the present invention relates to a method for detecting and / or quantifying MUC17 expression in a sample, the method comprising the following steps:

[0038] (a) Using an antibody generated according to any one of the foregoing embodiments or any of the foregoing embodiments, or using the detection system according to Example 15, to determine the expression level of MUC17 in a sample; and

[0039] (b) Compare the MUC17 expression level determined in step (a) with the following:

[0040] (c) Predefined values ​​for MUC17 expression levels

[0041] (d) The MUC17 expression level determined in the control sample, or

[0042] (e) MUC17 expression levels determined in samples obtained from the same source or subject at previous time points.

[0043] In the 22nd embodiment, the present invention relates to a method for diagnosing neoplastic diseases associated with MUC17 expression levels or increased MUC17 expression levels, the method comprising the following steps:

[0044] (a) Using an antibody generated according to any one of the embodiments or any one of the embodiments, or using the detection system according to Embodiment 15, to determine the expression level of MUC17 in a sample; and

[0045] (b) Compare the MUC17 expression level determined in step (a) with the following:

[0046] (c) A predefined cutoff value indicating the absence of this disease for MUC17 expression, or

[0047] (d) represents the level of MUC17 expression determined in negative control samples where this neoplastic disease is absent.

[0048] A higher level of MUC17 expression determined in step (a) compared to the predefined cutoff value in (i) or the MUC17 expression level determined in the negative control sample in (ii) indicates the presence of a disease associated with MUC17 expression or an increased level of MUC17 expression.

[0049] In the 23rd embodiment, the present invention relates to a method for monitoring the progression of a disease associated with MUC17 expression or increased MUC17 expression levels, or for monitoring the response to treatment for a disease associated with MUC17 expression or increased MUC17 expression levels, the method comprising the following steps:

[0050] (a) Using the antibody as described in any of the preceding embodiments, or using the detection system as described in Example 15, to determine the level of MUC17 expression in a sample obtained from a subject diagnosed with the disease at a first time point;

[0051] (b) Using the antibody as described in any of the foregoing embodiments, or using the detection system as described in Example 15, to determine the level of MUC17 expression in a sample obtained from the subject at a second time point or after treatment; and

[0052] (c) Compare the MUC17 expression level determined in step (a) with the MUC17 expression level determined in step (b);

[0053] A higher MUC17 expression level in step (a) compared to the MUC17 expression level determined in step (b) indicates that the disease is progressing, and / or a lower MUC17 expression level in step (a) compared to the MUC17 expression level determined in step (b) indicates that the disease is entering a remission phase or that the disease is responding to the treatment.

[0054] In the 24th embodiment, the present invention relates to the use or product as described in any one of Examples 16 to 20 or the method as described in any one of Examples 21 to 23, wherein the sample is a biological sample, preferably a human biological sample, such as a tissue sample or a sample containing cultured cells.

[0055] In the 25th embodiment, the present invention relates to the use or product as described in any one of Examples 16 to 20 or the method as described in any one of Examples 21 to 24, wherein the sample is obtained from a human subject, preferably a human subject suspected of having or having a disease associated with MUC17 expression or increased MUC17 expression, or a subject who has received treatment for a disease associated with MUC17 expression or increased MUC17 expression.

[0056] In the 26th embodiment, the present invention relates to the use or product as described in any one of Examples 16 to 20 or the method as described in any one of Examples 21 to 25, wherein the disease is selected from the group consisting of: esophageal cancer, gastric cancer, gastroesophageal cancer (including gastroesophageal junction cancer, GJC), gastrointestinal cancer, and pancreatic cancer.

[0057] In embodiment 27, the present invention relates to the use of any of the products as defined herein for detecting MUC17 expression in an immunohistochemical (IHC) method comprising the step of providing a tissue sample, including liquid biopsy material, such as cells obtained from an individual or animal or from a cell culture. The sample material is typically provided on a support, such as a carrier like a glass or plastic slide. The tissue can be fixed using fixation media known in the art, such as paraffin, ethanol, or acetone, or any other suitable medium that ensures fixation of the tissue or cells to the support. The sample is then prepared, for example, by deparaffining, and incubated with a medium that allows the antibodies described herein to approach the target (here, MUC17). The tissue is then incubated with a medium containing a sufficient amount of the antibodies disclosed herein to allow these antibodies to bind to MUC17 present in the tissue sample. After a generally known and reasonable amount of time, the incubation of the tissue with the antibodies is typically stopped by washing the support and tissue in a medium such as PBS. Both the incubation and washing steps can be performed at room temperature, but elevated or lower temperatures, such as from 2°C to 50°C, are possible. Typically, the incubation temperature depends on the incubation duration. For example, tissue can be stained overnight at 4°C, but the incubation time can also be shortened to a few minutes at higher temperatures, such as about 30°C to 40°C, for example, 37°C. To examine the effects of the incubation medium, fixation medium, incubation time, and temperature, positive control material with a known target expression (here, MUC17) and negative controls with samples known not to express MUC17 are typically used. Alternatively, (optionally, additionally) the antibody product of the present invention and blocking epitopes, i.e., epitopes corresponding to those specifically / selectively recognized or bound by the antibodies described herein, can also be used as negative controls. These blocking epitopes are added to the positive control material in a titration assay. The concentration of epitopes that prevent the antibodies of the present invention from specifically and / or selectively detecting MUC17 can thus be determined. In this way, the positive control—in the presence of blocking epitopes—no longer serves as a positive control because the amount of antibody binding MUC17 is blocked by a sufficient amount of blocking epitopes to saturate the antibody. Determining the correct amount of blocking epitopes and antibodies for appropriate positive and negative controls is known to those skilled in the art of immunocytochemistry, and particularly immunohistochemistry. Once appropriate controls are available, test materials, such as those obtained from patients suspected of having a disease characterized by pathologically enhanced / distributed MUC17 expression (e.g., cancer), can be analyzed. As discussed further below, antibodies may carry detectable markers, or they may be recognized by another conjugate carrying such a marker. The terminology for these techniques is known to those skilled in the art as direct immunocytology / immunohistochemistry detection and indirect immunocytology / immunohistochemistry detection.Once the required incubation and washing steps have been performed on the tissue samples and controls, the tissues are ready for analysis, for example, using an immunofluorescence-based detection system. The intensity and quantity of detectable markers can be determined and compared with positive and negative controls and / or with known standard values. Based on this, scientists are able to draw conclusions about whether a sample is positive for MUC17 expression. Attached Figure Description

[0058] Figures 1A to 1C Immunohistochemistry revealed high expression of MUC17 in gastric cancer. Immunohistochemistry using the antibody described herein revealed multifocal, diffuse MUC17 staining (brown staining) in human metastatic gastric cancer tissue sections. Figures 1A to 1B MUC17 expression is limited to the apical membrane of the normal human gastrointestinal tract (i.e., intestinal epithelial cells). Figure 1C ).

[0059] definition

[0060] Therefore, in one aspect, the present invention provides an antibody (or a portion or derivative thereof) that binds to MUC17. Hereinafter, whenever the term "antibody" (i.e., an antibody that binds to MUC17) is used, the term also implies that "antibody fragments" are included, which will be defined below. Furthermore, the definition and description of "antibody" (e.g., a monoclonal antibody that binds to MUC17) provided below applies similarly to any chemically or enzymatically modified antibody, such as an antibody carrying a label (e.g., a fluorescent, radioactive, luminescent, or chromogenic label) or an enzyme capable of generating a detectable signal. "Detectable signal" means that the label intensity on the MUC17 antibody can be measured using methods known in the art.

[0061] An "antibody" (sometimes also called an immunoglobulin) is a protein that binds specifically to its target. An antibody recognizes a unique target, known as an antigen, through its variable region. An "antibody" can be any immunoglobulin isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. The term "antibody" can include, for example, monoclonal antibodies, chimeric antibodies, recombinant antibodies, deimmunized antibodies, affinity-matured antibodies, humanized antibodies, and human antibodies, as well as antibodies from other species such as rodents, rabbits, mice, rats, hamsters, and goats. Antibodies can be derived from a single source or can be "chimeric," meaning that different parts of the antibody (such as the CDR, frame region, variable region, and constant region) can be derived from two different antibodies. The definition of "antibody" according to the invention includes full-length antibodies, as well as camel antibodies and other immunoglobulins generated through biotechnological or protein engineering methods or processes. Antibodies can also be produced in hybridomas.

[0062] A complete IgG antibody typically consists of two full-length heavy chains and two full-length light chains. The "light chain" comprises a variable region ("VL") with one domain and a constant region ("CL") with one domain. The variable region of the light chain is located at the amino terminus of the polypeptide. The light chain includes a κ chain and a λ chain. The "heavy chain" comprises a variable region ("VH") with one domain and a constant region ("CH") with—in the case of a complete IgG antibody—three domains: CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide.

[0063] In classic full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by a 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 heavy chain constant (CH) domain closest to the VH is usually named CH1. The constant (“C”) domain does not directly participate in antigen binding but exhibits various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation (complement-dependent cytotoxicity, CDC). The Fc region of an antibody is the “tail” region of a classic antibody, which interacts with cell surface receptors called Fc receptors and some proteins in the complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains (CH2 and CH3) of the two heavy chains of the antibody. The IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4) in each polypeptide chain. The Fc region also contains portions of a so-called "hinge" region held together by one or more disulfide bonds and non-covalent interactions. The Fc region of naturally occurring IgG has highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity.

[0064] The monoclonal antibody of the present invention is envisioned to be an IgG, IgD, IgE, IgM, or IgA antibody. According to one embodiment, the monoclonal antibody is an IgG antibody, such as IgG1, IgG2, IgG3, or IgG4. The isotype and subtype of the antibody may be mouse, rabbit, hamster, etc. (e.g., mouse IgG, mouse IgG1, etc.).

[0065] In the context of this invention, the term "variable" refers to those portions (i.e., "one or more variable regions") of an antibody or immunoglobulin domain that exhibit sequence variability and participate in determining the specificity and binding affinity of a particular antibody. Typically, the pairing of heavy chain variable regions (VH) and light chain variable regions (VL) together forms a single antigen-binding site. The variability is not uniformly distributed throughout the variable region of the antibody; it 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 region are called "framework" (FR) regions and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface. The variable regions of naturally occurring antibody heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4) that predominantly employ a β-sheet configuration. Together with the CDRs, they form the following sequence within the variable heavy or light chain: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Hypervariable regions in each chain are closely clustered together by framework regions and often contribute to the formation of antigen-binding sites together with hypervariable regions from another chain (see Kabat et al., Sequences of Proteins of Immunological Interest. Bethesda, National Institute of Health, 1991).

[0066] The term “CDR” and its plural “CDRs” refer to the complementary determinant regions of three binding features constituting the light chain variable regions (CDR-L1, CDR-L2, and CDR-L3) and three binding features constituting the heavy chain variable regions (CDR-H1, CDR-H2, and CDR-H3). CDRs contain most of the residues responsible for the specific interaction between the antibody (or antibody construct or binding domain) and the antigen, and thus contribute to the functional activity of the antibody molecule: they are the major determinants of antigen specificity. The precise definition of CDR boundaries and lengths is subject to different classification and numbering systems. Therefore, CDRs can be referred to by Kabat, Chothia, contact, or any other boundary definition (including the numbering system described herein). Despite the different boundaries, each of these systems has a degree of overlap in constituting the so-called “hypervariate regions” within the variable sequence. Therefore, CDR definitions according to these systems can differ in length and boundary regions relative to adjacent frame regions. See, for example, Kabat (a method based on cross-species sequence variability), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., cited above; 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 the AbM antibody modeling software from Oxford Molecular. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains in: Antibody Engineering Lab Manual (edited below: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Regarding the use of two residue identification techniques to define overlapping regions rather than identical regions, they can be combined to define heterozygous CDRs. However, numbering according to the so-called Kabat system is preferred.

[0067] Typically, CDR formation can be classified as canonical ring structures. The term "canonical structure" refers to the main chain conformation adopted by the antigen-binding (CDR) ring. From comparative structural studies, five of the six antigen-binding rings have been found to have only a limited library of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Thus, corresponding rings between antibodies can have very similar three-dimensional structures, despite high amino acid sequence variability in most parts of the ring (Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology], 1987, 196:901; Chothia et al., Nature [Nature], 1989, 342:877; Martin and Thornton, J. Mol. Biol. [Journal of Molecular Biology], 1996, 263:800). Furthermore, there is a relationship between the ring structure adopted and the surrounding amino acid sequence. The conformation of a particular canonical class is determined by the ring length and the amino acid residues located in key positions within the ring and within the conserved framework (i.e., outside the ring). Therefore, specific canonical categories can be assigned based on the presence of these key amino acid residues.

[0068] The term "canonical structure" can also include considerations regarding the linear sequence of the antibody, such as those cataloged by Kabat (Kabat et al., cited above). The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues of the variable region of an antibody in a consistent manner and is the preferred scheme for the application of this invention, as also mentioned elsewhere herein. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al. and / or revealed by other techniques, such as crystallography and two- or three-dimensional computational modeling. Thus, a given antibody sequence can be placed into a canonical category, which in particular allows for the identification of the appropriate category sequence (e.g., based on the expectation of including multiple canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations, as described by Chothia et al. (cited above), and their implications for interpreting canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known in the art. For a review of antibody structures, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, edited by Harlow et al., 1988.

[0069] The CDR3 of the light chain, and especially the CDR3 of the heavy chain, can constitute the most important determinants of antigen binding within the variable regions of both the light and heavy chains. In some antibodies or binding domains, the heavy chain CDR3 appears to constitute the primary contact region between the antigen and antibody. Altering the in vitro selection scheme of CDR3 alone can be used to alter the binding properties of the antibody / binding domain or to determine which residues contribute to antigen binding. Therefore, CDR3 is typically the largest source of molecular diversity within antibody binding sites. For example, CDR-H3 can be as short as two amino acid residues or longer than 26 amino acids.

[0070] The sequences of antibody genes are highly altered after assembly and somatic mutation, and it is estimated that these altered genes encode 10^10 different antibody molecules (Immunoglobulin Genes, 2nd ed., edited by Jonio et al., Academic Press, San Diego, CA, 1995). Therefore, the immune system provides an immunoglobulin repertoire. The term "repertoire" refers to at least one nucleotide sequence, wholly or partially derived from at least one sequence encoding at least one immunoglobulin. One or more sequences can be generated by in vivo rearrangement of the V, D, and J segments of the heavy chain and the V and J segments of the light chain. Alternatively, one or more sequences can be generated from cells in response to rearrangement (e.g., in vitro stimulation). Alternatively, a portion or all of one or more sequences can be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, for example, U.S. Patent 5,565,332. A repertoire may include only one sequence or may include multiple sequences, including sequences from a genetic diversity set.

[0071] The antibodies of this invention are envisioned to be monoclonal. As used herein, antibodies or binding domains designated as “monoclonal” (mAb) are obtained from a substantially homogeneous population of antibodies or binding domains, meaning that the individual antibodies or binding domains contained in the population are identical (specifically, regarding their amino acid sequences) except that they may be present in small amounts with naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Compared to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (or epitopes), monoclonal antibodies or binding domains exhibit high specificity against a single epitope within an antigen. In addition to their specificity, monoclonal antibodies also have the advantage of being synthesized via hybridoma cultures and thus not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the characteristic of antibodies or binding domains obtained from a substantially homogeneous population of antibodies or binding domains and should not be construed as requiring the antibody to be produced by any particular method.

[0072] To prepare monoclonal antibodies, any technique that provides antibodies produced through continuous cell line culture can be used. For example, the monoclonal antibody or binding domain to be used can be prepared by a hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Examples of other techniques for producing human monoclonal antibodies include the three-source hybridoma 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).

[0073] Then standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE) can be used. TM Hybridomas are analyzed and screened to identify one or more hybridomas that produce antibodies or binding domains that specifically bind to a specified antigen. Any form of the relevant antigen can be used as an immunogen, such as recombinant antigens, naturally occurring forms, chimeric antigens, any variants or fragments of that antigen, and its antigenic peptides. (See BIAcore for examples.) TM The surface plasmon resonance used in the system can be used to increase the efficiency of phage antibodies or binding domains that bind to epitopes of target antigens (Schier, Human Antibodies Hybridomas, 7 (1996), 97-105; Malmborg, J. Immunol. Methods, 183 (1995), 7-13).

[0074] Another exemplary method for preparing antibodies or binding domains 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. Patent 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).

[0075] In addition to using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (e.g., mice, hamsters, rabbits, or rats). In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, a large fragment of a human Ig (immunoglobulin) gene locus can be used to engineer mouse strains that are deficient in antibody production. By using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with desired specificity can be generated and selected. See, for example, Xenomouse. TM Green et al. (1994) Nature Genetics 7:13-21; US ​​2003-0070185; WO 96 / 34096 and WO 96 / 33735.

[0076] Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, such as humanization, deimmunization, rendering chimeric, etc. Examples of modified antibodies, constructs, or binding domains include humanized variants of non-human antibodies / antibody constructs, “affinity-matured” antibodies, constructs, or binding domains (see, for example, Hawkins et al. J. Mol. Biol. [Journal of Molecular Biology] 254, 889-896 (1992) and Lowman et al., Biochemistry [Biochemistry] 30, 10832-10837 (1991)), and antibody variants or mutants with one or more altered effector functions (see, for example, U.S. Patent 5,648,260; Kontermann and Dübel (2010), cited above; and Little (2009), cited above).

[0077] In immunology, affinity maturation is a process through which B cells produce antibodies with increased affinity for antigens during an immune response. Through repeated exposure to the same antigen, the host will produce antibodies with successively higher affinities. Similar to the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody fragments, antibody variants, or binding domains. Random mutations within the CDR are introduced using radiation, chemical mutagens, or error-prone PCR. Furthermore, genetic diversity can be increased through strand shuffling. Two or three rounds of mutation and selection using display methods (such as phage display) typically yield antibodies, antibody fragments, antibody variants, or binding domains with a low nanomolar affinity range.

[0078] Amino acid sequence modifications of the antibodies described herein have also been considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by peptide synthesis or by introducing appropriate nucleotide changes into the nucleic acid molecule encoding the antibody. All amino acid sequence modifications described below should produce antibodies that retain the desired biological activity of the unmodified parent molecule (binding to MUC17).

[0079] The term “amino acid” or “amino acid residue” typically refers to an amino acid having its recognized definition in the art, such as amino acids 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 (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as needed. There are essentially four different classes of amino acids, determined by different side chains:

[0080] (1) Nonpolar and neutral (uncharged): Ala, Gly, Ile, Leu, Met, Phe, Pro, Val

[0081] (2) Polar and neutral (uncharged): Asn, Cys (slightly polar only), Gln, Ser, Thr, Trp (slightly polar only), Tyr

[0082] (3) Acidity and polarity (negative charge): Asp and Glu

[0083] (4) Alkaline and polar (positively charged): Arg, His, Lys

[0084] Hydrophobic amino acids can be categorized based on whether they have aliphatic or aromatic side chains. Phe and Trp (very high hydrophobicity), Tyr and His (lower hydrophobicity) are classified as aromatic amino acids. Strictly speaking, aliphatic means that the side chain contains only hydrogen and carbon atoms. By this strict definition, amino acids with aliphatic side chains are alanine, isoleucine, leucine (also known as oroleucine), proline, and valine. Alanine has a very short side chain, meaning it is not particularly hydrophobic, and proline has an unusual geometry that allows it to play a special role in proteins. Methionine is often conveniently considered to be in the same category as isoleucine, leucine, and valine, although it also contains a sulfur atom. A unifying theme is that these amino acids primarily contain non-reactive and flexible side chains. Amino acids alanine, cysteine, glycine, proline, serine, and threonine are often grouped together because they are all quite small. Gly and pro can influence chain orientation.

[0085] Amino acid modifications include, for example, the deletion, insertion, and / or substitution of residues within the amino acid sequence of a monoclonal antibody or binding domain. Any combination of deletions, insertions, and / or substitutions may be performed to obtain the final monoclonal antibody or binding domain, provided that the final antibody possesses the desired characteristics, such as the biological activity of the unmodified parent molecule (e.g., binding to MUC17). Amino acid changes can also alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites.

[0086] For example, 1, 2, 3, 4, 5, or 6 amino acids can be inserted, deleted, and / or substituted in each CDR (depending on their respective lengths, of course), 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 can be inserted, deleted, and / or substituted in each FR. Amino acid sequence insertions also include N-terminal and / or C-terminal amino acid additions, ranging in length from, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing more than 10, for example, one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.

[0087] The sites of most interest for amino acid modifications (particularly amino acid substitutions) include hypervariable regions, particularly the individual CDRs of the heavy and / or light chains; however, FR variations in the heavy and / or light chains are also considered herein. Substitutions can be conservative substitutions as described herein. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted in the CDR, 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 can be substituted in the frame region (FR), depending on the length of the CDR or FR, respectively. For example, if the CDR sequence covers 6 amino acids, it is conceivable that one, two, or three of these amino acids will be substituted. Similarly, if the CDR sequence covers 15 amino acids, it is conceivable that one, two, three, four, five, or six of these amino acids will be substituted.

[0088] A useful method for identifying specific residues or regions within a monoclonal antibody or binding domain that serve as a preferred mutagenic site is known as “alanine scan mutagenesis,” and is described, for example, by Cunningham BC and Wells JA (Science. [Science] 1989 June 2; 244(4908):1081-5). Here, residues or groups of residues in the construct (e.g., charged residues such as Arg, His, Lys, Asp, and Glu) are identified and replaced with neutral or nonpolar amino acids (most preferably alanine or polyalanine) to influence the interaction of each amino acid with the target protein epitope. Alanine scan is a technique used to determine the contribution of specific residues to the stability or function of a given protein. Alanine is used because it has a non-giant, chemically inert methyl functional group, but still mimics the secondary structure preference of many other amino acids. In cases where the size of the conserved mutagenic residue is required, sometimes giant amino acids (such as valine or leucine) can be used. This technique can also be used to determine whether the side chains of specific residues play an important role in biological activity. Alanine scanning is typically performed via site-directed mutagenesis or randomization through the creation of PCR libraries. Furthermore, computational methods have been developed to estimate thermodynamic parameters based on theoretical alanine substitutions. Data can be obtained through IR / NMR spectroscopy, mathematical methods, bioassays, and other techniques.

[0089] Then, the amino acid positions exhibiting functional sensitivity to the substitution are precisely determined (e.g., by an alanine scan) by introducing additional or other variants at or to the substitution site. Thus, while the sites or regions used to introduce amino acid sequence changes are predetermined, 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, an alanine scan or random mutagenesis can be performed at the target codon or region, and the expressed monoclonal antibodies / variants can be screened for the optimal combination of desired activities. Techniques for substitution mutations at predetermined sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening for mutants is performed, for example, by an antigen binding activity assay using an antigen as described herein (e.g., MUC17).

[0090] Typically, if an amino acid is substituted in one or all of the CDRs in the heavy chain and / or light chain / variable region, it is envisioned that the resulting "substituted" sequence has at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identity / homology / similarity with the "original" or "parental" CDR sequence. This means that the degree of identity / homology / similarity between the original sequence and the substituted sequence depends on the length of the CDR. For example, a CDR with a total of 5 amino acids and containing one amino acid substitution has 80% identity with the "original" or "parental" CDR sequence, while a CDR with a total of 10 amino acids and containing one amino acid substitution has 90% identity with the "original" or "parental" CDR sequence. Therefore, the substituted CDRs of the monoclonal antibodies of the present invention can have different degrees of identity with their original sequences; for example, CDRL1 can have 80% homology, while CDRL3 can have 90% homology. The same considerations apply to the frame area and the entire VH and VL areas.

[0091] "Variant CDR" is a CDR that has specific sequence homology, similarity, or identity with the parental CDR of the present invention and shares biological functions with the parental CDR, 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 parental CDR. Typically, the amino acid homology, similarity, or identity among the various variant CDRs is at least 60% of the parental sequence described herein, and more typically has an increasing degree of homology, similarity, or identity of at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, and most preferably 95%, 96%, 97%, 98%, 99%, and almost 100%. This also applies to "variant VH" and "variant VL". According to one embodiment, sequence variations within "variant VH" and / or "variant VL" do not extend to the CDR. Therefore, the present invention relates to antibodies or derivatives or fragments as defined herein, which comprise VH and VL sequences having certain sequence homology / identity / similarity (see above) to specific sequences (“parental” VH and VL) as defined herein, wherein these CDR sequences are 100% identical to the specific CDR sequences (“parental” CDRs) as defined herein.

[0092] 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 1 below) is contemplated, provided that the monoclonal antibody (antibody construct) retains its ability to bind to MUC17, and / or as long as its CDR, FR, VH and / or VL sequences have at least 60% or 65%, more preferably at least 70% or 75%, even more preferably at least 80% or 85%, and particularly preferably at least 90% or 95% identity with the original or parental sequence.

[0093] Conservative substitution (also known as conserved mutation or conserved replacement) is an amino acid substitution that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, size). Conservative substitutions in proteins generally have a smaller impact on protein function compared to non-conservative substitutions. Conservative substitutions are illustrated in Table 1. Exemplary conservative substitutions are shown as "Exemplary Substitutions". If such substitutions result in changes in biological activity, further substantial changes, as described in detail below with reference to the amino acid categories, can be introduced, and desired characteristics of the product can be screened.

[0094] Table 1: Amino acid substitutions (aa = amino acid)

[0095]

[0096]

[0097] The substantial modification of the biological properties of the antibodies of the present invention is accomplished by selecting substitutions that are significantly different in maintaining the following effects: (a) the structure of the polypeptide backbone in the substituted region, such as folded or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the majority of the side chain. Non-conservative substitutions typically require exchanging a member of one of the amino acid classes defined above (such as polar, neutral, acidic, basic, aliphatic, aromatic, small, etc.) for another class. Any cysteine ​​residue that does not participate in maintaining the proper conformation of the antibody can often be substituted with serine to improve the oxidative stability of the antibody.

[0098] The sequence identity, homology, and / or similarity of amino acid sequences are determined using standard techniques known in the art, preferably using default settings or by testing. These standard techniques include, but are not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. [Advances in Applied Mathematics] 2:482; the sequence identity alignment algorithm of Needleman and Wunsch (J Mol Biol. [Journal of Molecular Biology] March 1970; 48(3):443-53); and the similarity search method of Pearson and Lipman (Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences of the United States of America] April 1988; 85(8):2444-8). The computerized implementations of these algorithms are GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin. Drive, Madison, Wis.), The best-fit sequence procedure described by Devereux et al. (Nucleic Acids Res. 11 Jan 1984; 12(1Pt 1):387-95). Percentage identity was envisioned to be calculated via FastDB based on the following parameters: mismatch penalty of 1; vacancy penalty of 1; vacancy size penalty of 0.33; and connection penalty of 30. See also “Current Methods in Sequence Comparison and Analysis”, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss.

[0099] A useful example of an algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create multiple sequence alignments from a set of related sequences. It can also plot dendrites that show the clustering relationships used to create the alignments. PILEUP uses a simplification of the progressive alignment method by Feng and Doolittle (J Mol Evol. [Journal of Molecular Evolution] 1987; 25(4):351-60); which is similar to the method described by Higgins and Sharp (Comput Appl Biosci. [Computers in the Biological Sciences] April 1989; 5(2):151-3). Useful PILEUP parameters include a default vacancy weight of 3.00, a default vacancy length weight of 0.10, and a weighted terminal vacancy.

[0100] Another example of a useful algorithm is the BLAST algorithm, described in the following: Altschul et al. (J MolBiol. [Journal of Molecular Biology] 10 Oct 5, 1990; 215(3):403-10.); Altschul et al. (Nucleic AcidsRes. [Nucleic Acids Research] 1 Sep 1, 1997; 25(17):3389-402); and Karlin and Altschul (Proc NatlAcad Sci US A. [Proceedings of the National Academy of Sciences] 15 June 1993; 90(12):5873-7). A particularly useful BLAST procedure is the WU-Blast-2 procedure, which is derived from Altschul et al. (Methods Enzymol. [Enzymological Methods] 1996; 266:460-80). WU-Blast-2 uses several search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap interval = 1, overlap score = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​and are determined by the program itself based on the composition of a specific sequence and a specific database used to search for sequences of interest; however, these values ​​can be adjusted to improve sensitivity.

[0101] Another useful algorithm is the gap BLAST, as reported by Altschul et al. (Nucleic Acids Res. 1 Sep 1, 1997; 25(17):3389-402). The gap BLAST uses BLOSUM-62 instead of scoring; the threshold parameter T is set to 9; a double-click method triggering non-gap expansion incurs a cost of 10+k for a gap length of k; Xu is set to 16, and Xg is set to 40 (for the database search phase) and 67 (for the algorithm's output phase). Alignments with gaps are triggered by scores corresponding to approximately 22 bits.

[0102] Consistent with this, the term "percentage of nucleic acid sequence identity / homology / similarity (%)" for the nucleic acid sequence encoding the monoclonal antibody (antibody construct) identified in this paper is defined as the percentage of nucleotide residues in the candidate sequence that are identical to those in the coding sequence of the antibody. One method for comparing two sequences and thus determining their homology is to use the BLASTN module of WU-Blast2 (set to default parameters), where the overlap span and overlap fraction are set to 1 and 0.125, respectively. Typically, the nucleotide sequence encoding each variant CDR shares at least 60% homology, similarity, or identity with the nucleotide sequence described herein, and more typically exhibits an increasing degree of homology, similarity, or identity of 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 almost 100%. Again, this also applies to the nucleotide sequences encoding “variant VH” and / or “variant VL”.

[0103] The term "antibody derivative" according to the invention may also comprise fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd(VH-CH1), heavy chain, Fab, Fab', F(ab')2, or "rIgG" (a "half-antibody" composed of a heavy chain and a light chain). Antibody fragments can be generated by enzymatic or chemical cleavage of a complete antibody. Antibody constructs according to the invention may also comprise modified fragments of the antibody, also referred to as antibody variants or antibody derivatives. Examples include, but are not limited to, scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, biantibodies, single-chain biantibodies, tandem biantibodies (Tandab), tandem di-scFv, tandem tri-scFv, “microantibodies” (which have structures such as (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multibody antibodies such as triabody or tetrabody, and single-domain antibodies (such as nanobodies or single variable domain antibodies containing only one variable region, which can be VHH, VH or VL, and which binds specifically to the antigen or target independently of other variable regions or domains). Other possible forms of antibody constructs according to the invention are cross-linked, maximal, hybrid Fc constructs, single Fc constructs, and scFc constructs. Examples of those forms will be described below. Furthermore, the term "antibody" is defined to include molecules consisting of only one polypeptide chain as well as molecules consisting of two, three, four, or more polypeptide chains, which may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). Examples of the antibodies identified above, their fragments, variants, derivatives, and binding domains derived therefrom, are particularly described in Harlow and Lane, Antibodies: A Laboratory Manual, CSHL Press (1988); Kontermann and Dübel, Antibody Engineering, Springer, 2nd edition, 2010; and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press, 2009.

[0104] The term "binding domain" or "domain that binds to" is used in connection with this invention to characterize a domain of an antibody that specifically binds to / interacts with / recognizes an epitope on a target or antigen (here: MUC17). The structure and function of the binding domain are based on the structure and / or function of the antibody, such as the structure and / or function of a full-length immunoglobulin molecule. Thus, a "binding domain" or "domain that binds to" may contain the minimum structural requirements of an antibody that allows for immune-specific target binding. This minimum structural requirement for a binding domain 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. A "domain that binds to" (or "binding domain") may typically contain both a variable region of the antibody light chain (VL) and a variable region of the antibody heavy chain (VH); however, it does not necessarily have to contain both, but may contain only one of VH or VL. For example, Fd fragments typically retain some antigen-binding functions of the complete antigen-binding domain.

[0105] Examples of “binding domain” or antibody constructs include, but are not limited to, full-length antibodies, fragments of full-length antibodies (such as VH, VHH, VL), (s)dAb, Fv, light chain (VL-CL), Fd(VH-CH1), heavy chain, Fab, Fab', F(ab')2, or “r IgG (“half-antibody”), antibody variants or derivatives (such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, biantibody, single-chain biantibody, tandem biantibody (Tandab), tandem di-scFv, tandem tri-scFv, “microantibody” (selected from forms such as (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3)), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multiantibody (such as triantibody or tetraantibody), and single-domain antibody (such as nanobody or single variable domain antibody) (containing only one variable region, which can be VHH, VH or VL). Other examples of the form of “binding domain” (or “binding domain”) include (1) antibody fragments or variants containing VL, VH, CL and CH1 (such as Fab); (2) antibody fragments or variants containing two linked Fab fragments (such as F(ab')2); (3) antibody fragments or variants containing VH and CH1 (such as Fd); (4) antibody fragments or variants containing VL and CL (such as light chains); (5) antibody fragments or variants containing VL and VH (such as Fv); (6) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546); (7) antibody variants containing at least three separate CDRs of heavy chains and / or light chains; and (8) single-chain Fv (scFv). Examples of embodiments of the antibodies according to the present invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO2014 / 151910, and WO 2015 / 048272.

[0106] In the scFv, the VH and VL regions are arranged in a VH-VL or VL-VH order (from the N-terminus to the C-terminus). It is envisioned that the VH and VL regions are connected via a linker (preferably a peptide linker). According to one embodiment, the VH region is located at the N-terminus of the linker, and the VL region is located at the C-terminus of the linker. Furthermore, it is possible that the two scFv domains of the antibody construct are connected via a linker (preferably a peptide linker). The scFv may, for example, contain domains in the order of first domain-linker-second domain (from the N-terminus to the C-terminus). A reverse order (second domain-linker-first domain) is also possible.

[0107] The linker is preferably a peptide linker, more preferably a short peptide linker. According to the invention, a "peptide linker" comprises an amino acid sequence that links an amino acid sequence of one domain of an antibody to another (variable and / or binding) domain (e.g., a variable domain or a binding domain). A key technical feature of such a peptide linker is that it does not contain any polymerization activity. Suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233 or WO 88 / 09344. In the context of the invention, a "short" linker has between 2 and 50 amino acids, preferably between 3 and 35, between 4 and 30, between 5 and 25, between 6 and 20, or between 6 and 17 amino acids. The linker between two variable regions of a binding domain may have a different length (e.g., it may be longer) than the linker between the two binding domains. For example, the linker between two variable regions of a binding domain can have a length of between 7 and 15 amino acids (preferably between 9 and 13), and the linker between two binding domains can have a length of between 3 and 10 amino acids (preferably between 4 and 8). Further, it is envisioned that the peptide linker is a glycine / serine linker. Most amino acids in the glycine / serine linker are selected from glycine and serine.

[0108] According to one embodiment of the invention, the antibody of the invention binding to MUC17 can be a “single-chain antibody.” Although the two domains VL and VH of the Fv fragment are encoded by independent genes, these two domains can be joined by an artificial linker using a recombinant approach, as described above, which allows them to be made into a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as full-length antibodies or IgG. Thus, a single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, typically linked by a short linker peptide. For flexibility, the linker is typically rich in glycine, and for solubility, it is typically rich in serine or also threonine, and can link the N-terminus of VH and the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin.

[0109] Antibodies named "single-domain antibodies" contain a (monomer) antibody variable region that can selectively bind to a specific antigen independently of other variable regions. The first single-domain antibodies were engineered from heavy-chain antibodies found in camels, and these are called VHH fragments. Cartilaginous fish also possess heavy-chain antibodies (IgNARs), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimer variable region from common immunoglobulins into monomers, thus obtaining VH or VL as single-domain Abs. Although most research on single-domain antibodies is currently based on heavy-chain variable regions, nanobodies derived from light chains have also shown specific binding to target epitopes. Examples of single-domain antibodies are so-called sdAbs, nanobodies, or single-variable-domain antibodies. Therefore, (single-domain mAb)2 is a monoclonal antibody composed of (at least) two single-domain monoclonal antibodies individually selected from the group containing VH, VL, VHH, and VNAR. The linker is preferably in the form of a peptide linker. Similarly, "scFv-single-domain mAb" is a monoclonal antibody consisting of at least one single-domain antibody as described above and an scFv molecule as described above. Likewise, the linker is preferably in the form of a peptide linker.

[0110] According to one embodiment, the antibody or antibody construct that binds to MUC17 is in the form of one or more peptides or in the form of a protein. In addition to the protein portion, such peptides or proteins may include non-protein portions (e.g., chemical linkers or chemical cross-linking agents such as glutaraldehyde).

[0111] Peptides are short chains of amino acid monomers linked by covalent peptide (amide) bonds. Therefore, peptides are classified as a broad chemical category of biological oligomers and polymers. The amino acids that form part of a peptide or polypeptide chain are called "residues" and can be numbered sequentially. All peptides, except cyclic peptides, have an N-terminal residue at one end and a C-terminal residue at the other. Oligopeptides consist of only a few amino acids (typically between two and twenty). Polypeptides are longer, continuous, and unbranched chains of peptides. Based on size, peptides differ from proteins; and as an arbitrary benchmark, a peptide can be understood as containing approximately 50 or fewer amino acids. Proteins consist of one or more polypeptides typically arranged in a manner consistent with their biological function. Although the aspects of laboratory techniques applied to peptides, polypeptides, and proteins differ (e.g., the characteristics of electrophoresis, chromatography, etc.), the size boundaries distinguishing peptides from polypeptides and proteins are not absolute. Therefore, in the context of this invention, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and the term "polypeptide" is generally preferred. The terms “peptide,” “polypeptide,” and “protein” also refer to naturally modified peptides / polypeptides / proteins, where modifications are achieved, for example, through post-translational modifications (such as glycosylation, acetylation, phosphorylation, etc.). When used herein, “peptide,” “polypeptide,” or “protein” can also refer to chemically modified peptides / polypeptides. Such modifications are well known in the art and are described below.

[0112] The terms “binds (specifically or immune-specifically) with,” “recognizes (specifically or immune-specifically) with,” or “reacts (specifically or immune-specifically) with” mean, according to the invention, that an antibody or binding domain interacts or (immuno-)specifically with a given epitope on a target molecule (antigen) (here: MUC17). Such interaction or binding occurs more frequently and rapidly at epitopes on a specific target than in alternative substances (non-target molecules), has a longer duration, greater affinity, or a combination thereof. However, due to sequence similarity between homologous proteins in different species, an antibody or binding domain that binds immune-specifically to its target (e.g., a human target) may cross-react with homologous target molecules from different species (e.g., from non-human primates, such as cynomolgus monkeys). Therefore, the term “specific / immunospecific binding” can include the binding of an antibody or binding domain to epitopes or structurally related epitopes in more than one species.

[0113] In the context of this invention, the term "epitope" refers to a portion or region of an antigen that is recognized / immunely specifically by a binding domain, antibody, or derivative thereof. An "epitope" is antigenic, and therefore the term is sometimes also referred to as an "antigen structure" or "antigen determinant." The portion of the binding domain, antibody, or antibody construct that binds to the epitope is called a paratope. Specific binding is believed to be achieved through a specific motif in the amino acid sequence of the binding domain, antibody, or antibody construct and the antigen. Thus, binding is achieved as a result of their primary, secondary, and / or tertiary structures and potential secondary modifications of said structures. Specific interactions between the paratope and its antigenic determinant can lead to simple binding of said site to the antigen. In some cases, specific interactions can alternatively or additionally lead to signal initiation, for example, due to induced changes in antigen conformation, antigen oligomerization, etc.

[0114] Based on the structure of epitopes and their interactions with complementary sites, protein antigen epitopes are classified into two categories: conformational epitopes and linear epitopes. Conformational epitopes consist of discontinuous portions of the antigen's amino acid sequence. These epitopes interact with complementary sites based on the antigen's three-dimensional surface features and shape or tertiary structure (folding). Methods for determining epitope conformation include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy. Conversely, linear epitopes interact with complementary sites based on their primary structure. Linear epitopes are formed from a continuous amino acid sequence derived from the antigen and typically contain at least 3 or at least 4, and more commonly at least 5 or at least 6 or at least 7, for example, about 8 to about 10 amino acids in a unique sequence.

[0115] The following describes a method for mapping the MUC17 epitope: a predefined region (typically a continuous amino acid extension) within the extracellular domain of the human MUC17 protein is exchanged / replaced with a corresponding region of MUC17 from another species (such as mouse, but other species can also be envisioned, provided the antibody does not cross-react with that species). These human MUC17 / mouse (or other species) MUC17 chimeras can be expressed on the surface of host cells (such as CHO cells). Binding to the antibody or antibody construct can be tested via FACS analysis. When the binding of the antibody to the chimeric molecule is completely eliminated, or when a significant reduction in binding is observed, it can be concluded that the region of human MUC17 removed from this chimeric molecule is associated with immune-specific epitope-complementary site recognition. Compared to binding to human (wild-type) MUC17, the reduction in binding 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%, thereby setting the binding to human MUC17 to 100%. Alternatively, the epitope mapping analysis described above can be modified by introducing one or more point mutations into the sequence of MUC17. These point mutations can, for example, reflect the differences between human MUC17 and mouse (or other species) MUC17.

[0116] Another method for determining the contribution of specific residues of a target antigen to antibody or binding domain recognition is an alanine scan (see, for example, Morrison KL and Weiss GA. Curr Opin Chem Biol. [New Insights in Chemical Biology] 2001 June; 5(3):302-7), in which each residue to be analyzed is replaced by alanine, for example, via site-directed mutagenesis. Alanine is used because it has a non-giant, chemically inert methyl functional group, but still mimics the secondary structure references of many other amino acids. In cases where the size of conserved mutant residues is required, sometimes giant amino acids (such as valine or leucine) can be used.

[0117] The interaction between a monoclonal antibody and the epitope of a target antigen means that the variable region exhibits a significant or marked affinity for the epitope / target antigen (here: MUC17) and generally does not exhibit a significant affinity for proteins or antigens other than the target antigen—although cross-reactivity with homologous targets from other species, as discussed above, is present. “Significant affinity” includes binding with an affinity (dissociation constant, KD) of about ≤10⁻⁶ M. Preferably, binding is considered specific when the binding affinity is about ≤10⁻⁷ M, ≤10⁻⁸ M, ≤10⁻⁹ M, or ≤10⁻¹⁰ M. Therefore, it is envisioned that the monoclonal antibody (or antibody construct) of the present invention has an affinity (KD) of about ≤10⁻⁷ M, ≤10⁻⁸ M, ≤10⁻⁹ M, or ≤10⁻¹⁰ M for MUC17. These values ​​are preferably measured in a surface plasmon resonance assay, such as a Biacore assay.

[0118] Whether an antibody reacts or binds to a target specifically (immuno-) can be readily tested, for example, by comparing the affinity of the antibody to its desired target protein or antigen with its affinity to a non-target protein or antigen (here: proteins other than MUC17). Preferably, the antibodies of the present invention do not bind significantly to proteins or antigens other than MUC17—unless one or more other binding domains targeting another target are intentionally introduced into the antibody / antibody construct of the present invention. The term “not significantly binding” means that the monoclonal antibody (or antibody construct) of the present invention does not bind to proteins or antigens other than MUC17. Thus, the antibody construct exhibits ≤30%, preferably ≤20%, more preferably ≤10%, particularly preferably ≤9%, ≤8%, ≤7%, ≤6%, ≤5%, ≤4%, ≤3%, ≤2%, or ≤1% reactivity with proteins or antigens other than MUC17, whereby the binding to MUC17 is set to 100%. “Reactivity” can be expressed, for example, as an affinity value (see above). It is envisioned that the monoclonal antibody (or derivative, binding domain, etc.) of the present invention does not bind to, interact with, recognize, immune-specifically bind to, or cross-react with human BAFF-R and / or human TACI.

[0119] The antibodies of this invention can be "in vitro generated antibodies" and / or "recombinant antibodies." In the context of this invention, the term "in vitro generated" refers to an antibody as defined above, wherein all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection process (e.g., in in vitro phage display, on a protein chip, or in any other method in which the ability of a candidate amino acid sequence to bind to an antigen can be tested). Therefore, this term preferably excludes sequences generated solely through genomic rearrangements in the immune cells of an animal. Antibodies are envisioned to be generated or available via phage display or library screening methods, or by transplanting a CDR sequence from a pre-existing antibody into a scaffold. "Recombinant antibody" is an antibody generated or produced using (especially) recombinant DNA technology or genetic engineering.

[0120] Preferred types of amino acid substitution changes in the antibodies of the present invention involve substituting one or more residues within a hypervariable region of the parent antibody structure. Typically, one or more resulting variants are selected for further development that will have improved biological properties relative to the parent antibody structure from which they are generated. A convenient method for generating such substituted variants includes phage-displayed affinity maturation. Briefly, several sites (e.g., 6-7 sites) of the hypervariable region are mutated to generate all possible amino acid substitutions at each site. The resulting variants are displayed monovalently from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened as disclosed herein. Alanine scanning mutagenesis can also be performed to identify candidate hypervariable sites (candidates for modification) that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the complex between the antigen and antibody or binding domain to identify contact points between the antibody binding domain and its specific antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, they are screened as described herein, and antibodies, their antigen-binding fragments, antibody constructs, or binding domains that exhibit superior properties in one or more relevant assays can be selected for further development.

[0121] According to one embodiment, the antibody is derived from a mouse. The term "antibody" includes, respectively, an antibody having antibody-derived regions such as variable and constant regions or domains substantially corresponding to germline immunoglobulin sequences known in the art. The binding domain of the present invention may contain, for example, amino acid residues in the CDR, and particularly CDR3, that are not encoded by the germline immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). The antibody binding domain may have at least one, two, three, four, five, or more sites substituted with amino acid residues not encoded by the germline immunoglobulin sequence. The definitions of antibody and binding domain as used herein also cover antibody and binding domains comprising only antibody sequences that are not artificially and / or genetically altered.

[0122] The antibodies of the present invention are envisioned as “isolated” or “substantially pure” antibodies. When used to describe the antibodies described herein, “isolated” or “substantially pure” means that the antibody has been identified, isolated, and / or recovered from the components of its production environment. Preferably, the antibody does not associate with, or substantially does not associate with, any other components from its production environment. Contaminating components of its production environment (such as those produced by recombinant transfected cells) are materials that can interfere with, for example, the diagnostic use of the antibody, and may include enzymes, hormones, and other protein or non-protein compounds. It should be understood that, depending on the circumstances, isolated or substantially pure antibodies can constitute from 5% to 99.9% by weight of the total protein / peptide content in a given sample. Using inducible or overexpressing promoters, the desired antibody can be produced at significantly higher concentrations. This definition includes the production of antibodies in a variety of organisms and / or host cells known in the art. In some embodiments, the antibody (1) is purified to a degree sufficient to obtain at least 15 N-terminal or internal amino acid residues using a spin-cup sequencer, or (2) is purified to homogenization by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. However, isolated antibodies are typically prepared by at least one purification step.

[0123] According to one embodiment of the present invention, the monoclonal antibody or binding domain comprises a VH region, which includes VH-CDR1 as depicted in SEQ ID NO:2, VH-CDR2 as depicted in SEQ ID NO:3, and VH-CDR3 as depicted in SEQ ID NO:4; and a VL region, which includes VL-CDR1 as depicted in SEQ ID NO:6, VL-CDR2 as depicted in SEQ ID NO:7, and VL-CDR3 as depicted in SEQ ID NO:8.

[0124] Further, it is envisioned that the antibody of the present invention binds to the same MUC17 epitope as the antibodies described in a), b), or c), or competes with the antibodies described in a), b), or c) for binding to MUC17.

[0125] Whether an antibody or binding domain binds to the same epitope of MUC17 (or the extracellular domain of MUC17) with another given antibody or binding domain can be measured by different analyses (e.g., by epitope mapping with chimeric or mutant MUC17 molecules, as described, for example, in WO 2013 / 072406). Other methods for determining epitopes are described in this paper, such as alanine scanning (see, for example, Morrison KL and Weiss GA. Curr Opin Chem Biol. [New Insights in Chemical Biology] 2001 June; 5(3):302-7), in which each residue within the target amino acid sequence to be analyzed is replaced by alanine, for example, via site-directed mutagenesis. Alanine is used because it has a non-giant, chemically inert methyl functional group, but still mimics the secondary structure references of many other amino acids. In cases where the size of conserved mutant residues is required, sometimes giant amino acids (such as valine or leucine) can be used. This method, which introduces systematic mutations of amino acids into the target protein sequence and tests the binding of antibodies to each mutant protein to identify the amino acids that make up the epitope, is also known as "site-directed mutagenesis." Other methods that can be used to map antibody epitopes on target antigens include high-throughput shotgun mutagenesis epitope mapping, cross-coupled mass spectrometry, X-ray cocrystallation, cryo-electron microscopy, and hydrogen-deuterium exchange.

[0126] Whether an antibody competes with another given antibody for binding to an antigen (such as MUC17) can be measured in competitive assays such as competitive ELISAs. Avidin-conjugated microparticles (beads) can also be used. Similar to avidin-coated ELISA plates, each of these beads can be used as a substrate on which an assay can be performed when reacted with a biotinylated protein. The antigen is coated onto the beads and then pre-coated with a first antibody. A second antibody is added and any additional binding is determined. Readings are generated via flow cytometry. Cell-based competitive assays can be used, using cells that naturally express MUC17 or cells stably or transiently transformed with MUC17. In the context of this document, the term “competitive binding” means at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% competition occurring between the two tested antibodies (as determined by any of the assays disclosed above).

[0127] According to one embodiment of the present invention, the antibody comprises:

[0128] a) The VH region contained in SEQ ID NO:5;

[0129] b) The VL region as depicted in any of SEQ ID NO:9;

[0130] Or the monoclonal antibody of the present invention

[0131] c) The antibody binds to the same MUC17 epitope as c) or competes with the antibody for binding to MUC17;

[0132] d) The antibody binds to the same MUC17 epitope as d) or competes with the antibody d) for binding to MUC17; or

[0133] e) The antibody binds to the same MUC17 epitope as e) or competes with the antibody for binding to MUC17.

[0134] The present invention envisions the monoclonal antibody (or “first monoclonal antibody”) and / or the second monoclonal antibody as defined herein binding to MUC17 in a sample. According to one embodiment, the sample may be a biological sample. According to one embodiment, the sample is a human sample, such as a human biological sample. The biological sample may be a (human) serum sample, plasma sample, blood sample, bone marrow sample, or tissue sample. The sample may also be a supernatant obtained from a cell culture of (human) bone marrow mononuclear cells or (human) peripheral blood mononuclear cells. The sample may be obtained from a subject, such as a person suspected of having or having (or diagnosed with) a disease associated with MUC17 or increased MUC17, or a subject who has received treatment for a disease associated with MUC17 or increased MUC17.

[0135] Blood is a bodily fluid in humans and other animals that delivers essential substances, such as nutrients and oxygen, to cells and removes metabolic waste products from these cells. In vertebrates, blood consists of blood cells suspended in plasma. Blood plasma, or "plasma," is the liquid component of blood in which several types of blood cells are suspended. It is primarily water and contains dissolved proteins (such as serum albumin, globulins, fibrinogen, etc.), glucose, clotting factors, electrolytes, hormones, carbon dioxide, and oxygen. Blood serum, or "serum," is plasma without clotting factors. Therefore, serum includes all plasma proteins not used for clotting. Bone marrow is a semi-solid tissue found within the spongy or reticular portion of bone. Tissue is the cellular level between cells and complete organs. Tissue is a collection of similar cells and their extracellular matrix from the same origin that together perform specific functions. Organs are then formed by functionally grouping multiple tissues together.

[0136] Covalent modification of the antibodies of this invention is also included within the scope of this invention and is generally, but not always, performed post-translational. For example, several types of covalent modification of the antibody can be introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that can react with selected side chains or with N-terminal or C-terminal residues. Derivatization with bifunctional agents can be used to crosslink the antibodies of this invention to a water-insoluble support matrix or surface for use in a variety of methods (particularly detection methods). Glutamine acyl and asparagine acyl residues are typically deamidated to their respective glutamine and asparagine residues. Alternatively, these residues are deamidated under mild acidic conditions. Any of these residue forms are within the scope of this invention. Other modifications include hydroxylation of proline and lysine, hydroxyphosphorylation of serine or threonyl residues, α-aminomethylation of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WHFreeman & Co., San Francisco, 1983, pp. 79-86), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0137] According to the present invention, monoclonal antibodies are conjugated to detectable tags. In some embodiments, covalent modification of the monoclonal antibodies of the present invention includes the addition of one or more tags, such as detection tags. Tags or tag groups can be conjugated to the antibody via spacer arms of various lengths to reduce potential steric hindrance. Various methods for tagging proteins are known in the art and can be used in carrying out the present invention. The term "tag" or "tag group" refers to any detectable tag. Generally, tags fall into several categories depending on the assay in which they will be detected—examples include, but are not limited to, the following:

[0138] a) Isotopic labeling, which may be radioactive or heavy isotopes, such as radioactive isotopes or radionuclides (e.g., 3 H, 14 C 15 N、 35 S, 89 Zr、 90 Y、 99 Tc, 111 In、 125 I, 131 I)

[0139] b) Magnetic markings (e.g., magnetic particles)

[0140] c) Redox-active components

[0141] d) Optical dyes (including, but not limited to, chromophores, phosphors, and fluorophores), such as fluorophores (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores, which may be "small molecule" fluorescent agents or protein fluorescent agents.

[0142] e) Enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase)

[0143] f) Biotinylated groups

[0144] g) Predetermined polypeptide epitopes recognized by the second reporter gene (e.g., leucine zipper pairs, binding sites of the second antibody, metal-binding domains, epitope tags, etc.).

[0145] "Fluorescent label" refers to any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, fluorescent yellow, waterfall 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), waterfall blue, waterfall yellow, and R-phycoerythrin (PE) (Molecular Probes Inc., Eugene, Oregon). 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 Richard P. Haugland's Molecular Probes Handbook.

[0146] Suitable protein fluorescent labeling also includes, but is not limited to, green fluorescent proteins, including GFP species such as Renilla, Ptilosarcus, or Aequorea (Chalfie et al., 1994, Science 263:802-805), and EGFP (Clontech Laboratories, Inc.). Accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9); Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.) Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. [Journal of Immunology] 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85:2603-2607) and Renilla (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, US Patent 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; 5,925,558).

[0147] The antibodies of the present invention may also contain additional domains, such as those that facilitate the separation of molecules. Domains facilitating antibody separation may be selected from peptide motifs or auxiliaryly introduced portions that can be captured in separation methods (e.g., separation columns). Non-limiting examples of such additional domains include peptide motifs called Myc-tag, HAT-tag, HA-tag, TAP-tag, GST-tag, chitin-binding domain (CBD-tag), maltose-binding protein (MBP-tag), Flag-tag, Strep-tag, and variants thereof (e.g., StrepII-tag), and His-tag. All antibodies disclosed herein characterized by an identified CDR may contain a His-tag domain, which is generally referred to as a repeat of consecutive His residues in the amino acid sequence of the molecule, such as a repeat of five or six His residues (hexahistidine). The His-tag may be located, for example, at the N-terminus or C-terminus of the antibody. In one embodiment, the hexahistidine tag is linked to the C-terminus of the antibody according to the invention via a peptide bond.

[0148] This invention further provides a polynucleotide / nucleic acid molecule encoding the antibody of the present invention. Nucleic acid molecules are biopolymers composed of nucleotides. Polynucleotides are biopolymers composed of 13 or more nucleotide monomers covalently bonded in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotide / nucleic acid molecules with different biological functions. Nucleotides are organic molecules that act as monomers or subunits of nucleic acid molecules such as DNA or RNA. The nucleic acid molecule or polynucleotide of the present invention can be double-stranded or single-stranded, linear or circular. It is envisioned that the nucleic acid molecule or polynucleotide is contained in a vector. Furthermore, it is envisioned that such a vector is contained in a host cell. The host cell is able to express the antibody, for example, after transformation or transfection with the vector or polynucleotide / nucleic acid molecule of the present invention. For this purpose, the polynucleotide or nucleic acid molecule is operatively linked to a control sequence.

[0149] The genetic code is a set of rules that translates the information encoded within genetic material (nucleic acids) into proteins. Biological decoding in living cells is accomplished by ribosomes linking amino acids in a sequence specified by mRNA, using tRNA molecules to carry the amino acids and read them out as three nucleotides at a time. This code defines how the sequence of these nucleotide triplets (called codons) specifies which amino acid will be added next during protein synthesis. With some exceptions, trinucleotide codons in nucleic acid sequences specify a single amino acid. Because most genes are encoded using essentially the same code, this particular code is often referred to as the canonical or standard genetic code.

[0150] Codon degeneracy is the redundancy of the genetic code, manifested as the multiplicity of three-base pair codon combinations that specify an amino acid. Degeneracy occurs because there are more codons than can encode an amino acid. A codon encoding an amino acid can differ in any of its three positions; however, this difference is usually at the second or third position. For example, codons GAA and GAG both specify glutamic acid and exhibit redundancy; however, neither specifies any other amino acid and therefore does not exhibit ambiguity. The genetic code of different organisms may be biased towards using one of several codons encoding the same amino acid rather than others—that is, one that will be found to have a greater frequency than would be expected by chance. For example, leucine is specified by six different codons, some of which are rarely used. Codon usage tables detailing the frequency of codon usage in the genomes of most organisms are available. Recombinant gene technology typically leverages this effect by implementing a technique called codon optimization, in which those codons are used to design polynucleotides preferred by their respective host cells (such as human hamster-derived cells, Escherichia coli cells, or Saccharomyces cerevisiae cells), for example, to increase protein expression. Therefore, it is contemplated that the polynucleotide / nucleic acid molecules disclosed herein are codon-optimized. However, any codon encoding the desired amino acid can be used to design polynucleotide / nucleic acid molecules encoding the antibodies of this invention.

[0151] According to one embodiment, the polynucleotide / nucleic acid molecule of the present invention encoding the antibody of the present invention is in the form of a single molecule or two or more separate molecules. If the antibody construct of the present invention is single-chain, the polynucleotide / nucleic acid molecule encoding such a construct will most likely also be in the form of a single molecule. However, it is also contemplated that the different components of the antibody (such as the heavy chain and light chain) are located on separate polypeptide chains, in which case the polynucleotide / nucleic acid molecule is most likely to be in the form of two (or more) separate molecules.

[0152] This also applies to vectors containing the polynucleotide / nucleic acid molecules of the present invention. If the antibody of the present invention is a single-chain antibody, a vector may contain a polynucleotide encoding the antibody at a single location (as a single open reading frame, ORF). A vector may also contain two or more polynucleotide / nucleic acid molecules at separate locations (with separate ORFs), each encoding a different component of the antibody of the present invention (such as heavy and light chains). It is envisioned that vectors containing the polynucleotide / nucleic acid molecules of the present invention may be in the form of a single vector or in the form of two or more separate vectors. In one embodiment, and for the purpose of expressing the antibody in a host cell, the host cell of the present invention should contain either a polynucleotide / nucleic acid molecule encoding the antibody or all of a vector containing such a polynucleotide / nucleic acid molecule, meaning that all components of the antibody—whether encoded as a single molecule or encoded at separate molecules / locations—will assemble together after translation to form the bioactive antibody of the present invention.

[0153] This invention also provides vectors comprising the polynucleotide / nucleic acid molecules of this invention. A vector is a nucleic acid molecule used as a medium for transferring (foreign) genetic material into cells, typically for replication and / or expression. The term "vector" encompasses, but is not limited to, plasmids, viruses, granules, and artificial chromosomes. Some vectors are specifically designed for cloning (cloning vectors), while others are designed for protein expression (expression vectors). So-called transcription vectors are primarily used to amplify their inserts. DNA manipulation is typically performed on E. coli vectors containing the elements necessary for their maintenance in E. coli. However, vectors can also have elements that allow them to be maintained in another organism, such as yeast, plant, or mammalian cells, and these vectors are called shuttle vectors. Inserting a vector into a target or host cell is generally referred to as transformation (for bacterial cells) and transfection (for eukaryotic cells), while the insertion of a viral vector is generally referred to as transduction.

[0154] Generally, engineered vectors contain an origin of replication, a multiple cloning site, and a selection marker. The vector itself is typically a nucleotide sequence (usually a DNA sequence) containing an insert (transgenic gene) and a larger sequence that acts as the vector's "backbone." While the genetic code determines the polypeptide sequence of a given coding region, other genomic regions can influence the timing and location of these polypeptide production. Therefore, modern vectors can encompass additional features beyond the transgenic insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, target sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are particularly used for expressing transgenes in target cells and typically contain control sequences.

[0155] The term "control sequence" refers to the DNA sequence necessary for the expression of an operablely linked coding sequence in a host organism. For example, control sequences suitable for prokaryotes include promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, Kozak sequences, and enhancers.

[0156] When a nucleic acid is functionally related to another nucleic acid sequence, that nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is localized to promote translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked nucleotide sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and in the reading phase. However, enhancers do not need to be contiguous. Linkage is accomplished by joining at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0157] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. This term is primarily used for non-viral methods in eukaryotic cells. Transduction is generally used to describe the transfer of nucleic acid molecules or polynucleotides mediated by viruses. Transfection in animal cells typically involves opening transient pores or "holes" in the cell membrane to allow for the uptake of substances. Transfection can be performed using biological particles (such as viral transfection, also known as viral transduction), chemical-based methods (such as using calcium phosphate, lipid transfection, Fugene, cationic polymers, nanoparticles), or physical treatments (such as electroporation, microinjection, gene gun, cell squeezing, magnetic transfection, hydrostatic pressure, impalefection, ultrasound, optical transfection, and heat shock).

[0158] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and into non-animal eukaryotic cells (including plant cells). Therefore, transformation is a genetic alteration in bacteria or non-animal eukaryotic cells that results from the direct uptake of exogenous genetic material (nucleic acid molecules) from their surroundings via one or more cell membranes and subsequent incorporation. Transformation can be performed artificially. For transformation to occur, the cell or bacteria must be in a competent state, which may occur as a time-limited response to environmental conditions such as starvation and cell density, and can also be induced artificially.

[0159] Furthermore, this invention provides a host cell that has been transformed or transfected by the polynucleotide / nucleic acid molecule of this invention or the vector of this invention. As used herein, the terms "host cell" or "recipient cell" are intended to include any single cell or cell culture that may be or is already a receptor for a vector, exogenous nucleic acid molecule, and / or a polynucleotide encoding an antibody of this invention, and / or a receptor for the antibody itself. The corresponding material is introduced into the cell by transformation, transfection, etc. (see above). The term "host cell" is also intended to include the offspring or potential offspring of a single cell. Because certain changes may occur in subsequent generations due to natural, accidental, or intentional mutations or due to environmental influences, such offspring may not actually be completely identical to the parent cell (in morphology, genome, or complete DNA complement), but are still included within the scope of the terminology used herein. Suitable host cells include prokaryotic or eukaryotic cells, and include, but are not limited to, bacteria (such as Escherichia coli), yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells (e.g., hamster, rodent, rat, macaque, or human cells).

[0160] Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are suitable cloning or expression hosts for the antibodies of this invention. Saccharomyces cerevisiae or Bacillus oryzae are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and can be used in this paper, such as *Schizosaccharomyces pombe*, hosts of *Kluyveromyce* such as *Kluyveromyce* lactis, *Kluyveromyce* fragilis (ATCC 12424), *Kluyveromyce* bulgaricus (ATCC 16045), *Kluyveromyce* wickeramii (ATCC 24178), *Kluyveromyce* waltii (ATCC 56500), *Kluyveromyce* drosophilarum (ATCC 36906), *Kluyveromyce* thermotonicus, and *Kluyveromyce* Marxianus; *Yersinia* (EP 402226); *Pichia pastoris* (EP 183). 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as Aspergillus nidulans and Aspergillus niger.

[0161] Suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants from hosts such as the fall armyworm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm), as well as corresponding permitted insect host cells, have been identified. Many viral strains used for transfection are publicly available, such as the L-1 variant of the alfalfa silver-striped armyworm (Autographa californica) NPV and the Bm-5 strain of the silkworm NPV, and such viruses can be used as the viruses described herein, particularly for transfecting fall armyworm cells.

[0162] Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors that can be used to produce 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 Journal 8:745-750, and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0163] However, the greatest interest is in vertebrate cells, and the propagation of vertebrate cells in cultures (cell cultures) has become a routine procedure. Examples of useful mammalian host cell lines include monkey kidney CV1 lines transformed from SV40 (such as COS-7, ATCC CRL1651); human embryonic kidney lines (such as 293 cells or subclones used for growth in suspension culture, Graham et al., J. Gen Virol. [Journal of General Virology] 36:59 (1977)); juvenile hamster kidney cells (such as BHK, ATCC CCL 10); Chinese hamster ovary / -DHFR cells (such as CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 77:4216 (1980)); mouse setoli cells (such as TM4, Mather, Biol. Reprod. [Reproductive Biology] 23:243-251 (1980)); monkey kidney cells (such as CVI ATCC CCL 70); and African green monkey kidney cells (such as VERO-76, ATCC). CRL1587); human cervical cancer cells (such as HELA, ATCC CCL 2); canine kidney cells (such as MDCK, ATCC CCL 34); Buffalo rat hepatocytes (such as BRL 3A, ATCC CRL 1442); human lung cells (such as W138, ATCC CCL 75); human hepatocytes (such as Hep G2, 14138065); mouse mammary tumors (such as MMT 060562, ATCC CCL-51); TRI cells (Mather et al., Annals N. YAcad. Sci. [Annals of the New York Academy of Sciences] (1982) 383: 44-68); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell lines (such as Hep G2).

[0164] In another embodiment, the present invention provides a process for generating the antibodies of the present invention, the process comprising culturing host cells of the present invention under conditions that allow expression of the antibodies of the present invention and recovering the generated antibodies from the culture.

[0165] As used herein, the term "culture" refers to the in vitro maintenance, differentiation, growth, proliferation, and / or reproduction of cells in a culture medium under suitable conditions. This involves growing and maintaining cells in a cell growth medium at appropriate temperatures and gas mixtures. Culture conditions vary considerably for each cell type. Typical growth conditions are a temperature of approximately 37°C, a CO2 concentration of approximately 5%, and a humidity of approximately 95%. The formulation of growth media can vary, for example, in terms of pH, the concentration of carbon sources (such as glucose), the nature and concentration of growth factors, and the presence of other nutrients (such as amino acids or vitamins). Growth factors used to supplement the culture medium are often derived from animal blood serum, such as fetal bovine serum (FBS), calf serum (FCS), horse serum, and porcine serum. Cells can be grown in suspension or as adherent cultures. Cell lines that have been modified to survive in suspension cultures also exist, allowing them to grow at higher densities than would be permitted under adherent conditions.

[0166] The term "expression" includes any step involved in generating the antibodies of the present invention, including but not limited to transcription, post-transcriptional modification, translation, folding, post-translational modification, targeting of specific subcellular or extracellular sites, and secretion. The term "recovery" refers to a series of processes aimed at isolating antibodies from cell cultures. "Recovery" or "purification" processes can separate the protein and non-protein fractions of a cell culture and ultimately isolate the desired antibody from all other peptides and proteins. Isolation steps typically utilize differences in protein size, physicochemical properties, binding affinity, and biological activity. Preparative purification aims to produce relatively large quantities of purified protein for subsequent use, while analytical purification produces relatively small quantities of protein for various research or analytical purposes.

[0167] When using recombinant technology, antibodies can be generated intracellularly in the periplasmic space or secreted directly into the culture medium. If the antibody is generated intracellularly, particulate fragments of the host cell or lysate are removed as a first step, for example, by centrifugation or ultrafiltration. The antibodies of the present invention can be generated, for example, in bacteria such as *Escherichia coli*. After expression, the construct is separated from the bacterial cell paste in the soluble fraction and can be purified, for example, by affinity chromatography and / or size exclusion. The final purification can be performed in a manner similar to that used for purifying antibodies expressed in mammalian cells and secreted into the culture medium. Carter et al. (Biotechnology (NY) Feb 1992; 10(2):163-7) describe a procedure for isolating antibodies secreted into the periplasmic space of *E. coli*.

[0168] In cases where antibodies are secreted into the culture medium, the supernatant from such expression systems is typically concentrated first using a commercially available protein concentrator (e.g., an ultrafiltration unit).

[0169] The antibodies 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 to be recovered, other techniques for protein purification are also available, such as fractionation on ion exchange columns, mixed-mode ion exchange, HIC, ethanol precipitation, size exclusion chromatography, reversed-phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), immunoaffinity (such as protein A / G / L) chromatography, chromatographic focusing, SDS-PAGE, ultracentrifugation, and ammonium sulfate precipitation. Protease inhibitors may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of contaminants.

[0170] Furthermore, the present invention provides a composition or formulation comprising the antibody of the present invention or comprising an antibody produced by a process according to the present invention. The composition is preferably a diagnostic composition. As used herein, the term "diagnostic composition" refers to a composition suitable for use in a diagnostic kit or detection system. One possible diagnostic composition of the present invention comprises one or more antibodies of the present invention, preferably in an amount suitable for detecting MUC17 in a sample. The diagnostic composition may further comprise suitable formulations of one or more loaders, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, and / or adjuvants. The diagnostic compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0171] The composition may contain a carrier, such as a diagnostically acceptable carrier. Generally, as used herein, “diagnostologically acceptable carrier” means all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate-buffered saline (PBS) solutions), water, suspensions, emulsions (e.g., oil / water emulsions), various types of wetting agents, liposomes, dispersion media, and coatings compatible with diagnostic applications. The use of such media and agents in diagnostic compositions is well known in the art, and compositions containing such carriers can be formulated using well-known conventional methods.

[0172] Some embodiments provide diagnostic compositions comprising the antibodies of the present invention and one or more additional excipients, such as those illustratively described in this section and elsewhere herein. Excipients may be used in the present invention for a variety of purposes, such as adjusting the physical, chemical, or biological properties of formulations, such as adjusting viscosity and / or the processes of the present invention to improve efficacy and / or stabilize such formulations and processes to prevent degradation and spoilage, for example, due to stress occurring during manufacturing, transport, storage, preparation before use, application, and thereafter. Excipients should generally be used at their lowest effective concentration.

[0173] In some embodiments, the diagnostic composition may contain formulation materials (see Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing Company) to modify, maintain, or preserve certain characteristics of the composition (such as pH, molar osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability). In such embodiments, suitable formulation materials may include, but are not limited to:

[0174] amino acids

[0175] • Antimicrobial agents, such as antibacterial agents and antifungal agents

[0176] Antioxidants

[0177] • Buffer solutions, buffer systems, and buffering agents for maintaining compositions at physiological pH or slightly lower (typically in the pH range of about 5 to about 8 or 9).

[0178] • Non-aqueous solvents, vegetable oils, and injectable organic esters

[0179] • Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline solutions and buffer media.

[0180] • Biodegradable polymers, such as polyester

[0181] • Accumulation agent

[0182] · Chelating agents

[0183] Isotonic agents and absorption delay agents

[0184] Complexing agents

[0185] • Filler

[0186] ·carbohydrate

[0187] • (Low molecular weight) proteins, peptides, or protein carriers, preferably of human origin.

[0188] Coloring agents and flavoring agents

[0189] • Sulfur-containing reducing agents

[0190] • Diluent

[0191] Emulsifier

[0192] Hydrophilic polymers

[0193] Salt formation counterions

[0194] ·preservative

[0195] Metal complexes

[0196] Solvents and cosolvents

[0197] Sugars and sugar alcohols

[0198] ·Suspension

[0199] Surfactants or wetting agents

[0200] • Stability enhancer

[0201] Tension enhancer

[0202] Parenteral delivery media

[0203] Intravenous delivery of mediators

[0204] It is well known that different components of a diagnostic composition can have different effects; for example, amino acids can act as buffers, stabilizers and / or antioxidants; mannitol can act as build-up agents and / or tension enhancers; sodium chloride can act as a delivery medium and / or tension enhancer; and so on.

[0205] According to another aspect, the present invention provides a detection system comprising: a) a first monoclonal antibody (or a derivative thereof) that binds to MUC17, and b) a second monoclonal antibody (or a derivative thereof) that optionally binds to MUC17, wherein the binding of the first monoclonal antibody (or a derivative thereof) to MUC17 occurs in the presence of the second monoclonal antibody (or a derivative thereof) that binds to MUC17.

[0206] The present invention also provides a detection system comprising: a) a first monoclonal antibody (or a derivative thereof) that binds to MUC17, and b) a second monoclonal antibody (or a derivative thereof) that optionally binds to MUC17, wherein the binding of the second monoclonal antibody (or a derivative thereof) to MUC17 occurs in the presence of the first monoclonal antibody (or a derivative thereof) that binds to MUC17.

[0207] The present invention also provides a detection system comprising: a) a first monoclonal antibody (or a derivative thereof) that binds to MUC17, and b) a second monoclonal antibody (or a derivative thereof) optionally that binds to MUC17, wherein the binding of the first monoclonal antibody (or a derivative thereof) to MUC17 occurs in the presence of the second monoclonal antibody (or a derivative thereof) that binds to MUC17, and wherein the binding of the second monoclonal antibody (or a derivative thereof) to MUC17 occurs in the presence of the first monoclonal antibody (or a derivative thereof) that binds to MUC17.

[0208] A "detection system" is a kit or tool (or diagnostic kit / tool) containing reagents for performing an analytical assay. In the context of this invention, the assay detects and / or quantifies the presence of MUC17 in a sample (typically a tissue sample). The detection system contains an antibody that binds to MUC17. Typically, the detection system involves using a solid support (such as a microtiter plate or membrane) that acts as a surface to immobilize the antigen to be detected (e.g., in the case of a "direct ELISA") or a (monoclonal) antibody ("capture antibody") that binds to MUC17, or a "second antibody" (e.g., an anti-Fc antibody) that binds to the antibody that binds to MUC17 (capture antibody). Typically, this immobilization occurs nonspecifically (by adsorption onto a surface) or specifically (by capture by an antibody, such as a second antibody). In addition, the detection system may include a (monoclonal) detection antibody that binds to MUC17 (optionally coupled to an enzyme, a detectable label, or a reporter group), and optionally a second antibody (e.g., an anti-Fc antibody) that binds to the detection antibody and is coupled to the enzyme, the detectable label, or the reporter group.

[0209] A well-known detection system is the ELISA assay, which can be used for the purposes of this invention. A “sandwich” ELISA is used to detect a sample antigen or to quantify an unknown amount of antigen. These steps may include: providing a surface for binding to a known amount of “capture antibody.” This binding can occur directly by adsorbing the capture antibody onto the surface, or by a second antibody (e.g., an anti-Fc antibody) adsorbed onto the surface and bound to the capture antibody. Any non-specific binding sites on the surface are blocked. A sample containing the antigen is applied to the surface, and the antigen is captured (bound) by the antibody. The plate is washed to remove unbound antigen. A “detection antibody” is added, and the “detection antibody” binds to the antigen. This detection antibody may be coupled (e.g., covalently linked) to an enzyme, a detectable label, or a reporter group. If this is not the case, a second antibody coupled to an enzyme, a detectable label, or a reporter group and bound to the detection antibody (e.g., to its Fc region) is applied. The plate is washed to remove any unbound antibody. A chemical substrate is added, which is converted (e.g., by an enzyme) into a detectable form, such as an optical signal (e.g., color or fluorescence) or an electrochemical signal. Measure the absorbance, fluorescence, or electrochemical signal (e.g., current) of the plate wells or surface to determine the presence and / or amount of antigen. Commonly used enzyme markers include:

[0210] -OPD (o-phenylenediamine dihydrochloride) turns amber to detect horseradish peroxidase (HRP), an enzyme commonly used as a conjugated protein.

[0211] -TMB (3,3',5,5'-tetramethylbenzidine) turns blue when detecting HRP and yellow when detecting sulfuric acid or phosphoric acid.

[0212] - When HRP is detected, ABTS (2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) turns green.

[0213] - When testing for alkaline phosphatase, PNPP (disodium p-nitrophenyl phosphate) turns yellow.

[0214] Traditional ELISA typically involves chromogenic reporter molecules and substrates, which produce observable color changes to indicate the presence of an antigen. Newer ELISA-like techniques use fluorescent, electrochemiluminescent, and quantitative PCR reporter molecules to generate quantifiable signals. These new reporter molecules can offer several advantages, including higher sensitivity and multiplexing. Technically, these assays are not strictly "ELISAs" because they are not "enzyme-linked" but rather linked to some non-enzymatic reporter molecule. However, because the general principles underlying these assays are largely similar, they are often categorized together with ELISAs.

[0215] This detection system can be used qualitatively or quantitatively. Qualitative results provide a simple positive or negative (yes or no) result for the sample. The cutoff between positive and negative may be statistical. Typically, two or three times the standard deviation (the inherent error of the test) is used to distinguish between positive and negative samples. Quantitatively, the optical density (OD) or electrochemical signal of the sample is compared to a standard curve, typically a series of dilutions of a target molecule solution of known concentrations.

[0216] This invention also provides a first monoclonal antibody (or binding domain) for the detection system:

[0217] a) Contains a VH region, which includes VH-CDR1 as depicted in SEQ ID NO:2, VH-CDR2 as depicted in SEQ ID NO:3, and VH-CDR3 as depicted in SEQ ID NO:4; and a VL region, which includes VL-CDR1 as depicted in SEQ ID NO:6, VL-CDR2 as depicted in SEQ ID NO:7, and VL-CDR3 as depicted in SEQ ID NO:8;

[0218] b) The antibody binds to the same MUC17 epitope as the antibody in a), or competes with the antibody in a) for binding to MUC17; or

[0219] c) The antibody binds to the same MUC17 epitope as b) or competes with the antibody for binding to MUC17.

[0220] This invention also provides monoclonal antibodies for the detection system:

[0221] a) Contains the VH region included in SEQ ID NO:5;

[0222] b) Contains the VL region included in SEQ ID NO:7;

[0223] c) Includes the VH region contained in SEQ ID NO:5 and the VL region contained in SEQ ID NO:7;

[0224] d) The antibody binds to the same MUC17 epitope as c) or competes with the antibody in c) for binding to MUC17; or

[0225] e) The antibody binds to the same MUC17 epitope as d) or competes with the antibody of d) for binding to MUC17.

[0226] Imagine a detection system that uses a first monoclonal antibody as the capture antibody and a second monoclonal antibody as the detection antibody.

[0227] In another aspect, the present invention also provides the use of the antibodies of the present invention in the following aspects or the use of the detection system of the present invention in the following aspects:

[0228] - Detect MUC17 in the sample;

[0229] -Quantitative analysis of MUC17 in the sample;

[0230] - Diagnose diseases associated with MUC17 or increased MUC17;

[0231] - Stratify patients diagnosed with diseases associated with MUC17 or increased MUC17;

[0232] - Monitor the progression of diseases associated with MUC17 or increased MUC17; or

[0233] - Monitor the response to treatment for diseases associated with MUC17 or increased MUC17.

[0234] In one embodiment, the sample is a biological sample, such as a human biological sample. The sample (biological sample / human biological sample) can be a serum sample, plasma sample, blood sample, bone marrow sample, or tissue sample. The sample can also be a supernatant obtained from a cell culture of bone marrow mononuclear cells or peripheral blood mononuclear cells. The sample can be obtained from a subject, such as a subject suspected of having or diagnosed with a disease associated with MUC17 or increased MUC17, or a subject who has received treatment for a disease associated with MUC17 or increased MUC17. In a particular embodiment, the sample is a tissue sample derived from tissue suspected of being affected by neoplastic growth (i.e., cancer).

[0235] A disease is a specific abnormal condition that negatively affects part or all of the structure or function of an organism (such as a human) without being caused by any external injury. Diseases are generally interpreted as a “medical condition” or “disorder” associated with specific signs and symptoms. According to the invention, a disease is associated with MUC17 or increased MUC17. The term “increased” is used in comparison to healthy subjects (i.e., subjects without the disease). According to one embodiment, a disease associated with MUC17 or increased MUC17 is a “MUC17-positive vegetation.”

[0236] "Vessence" is an abnormal growth of tissue that usually, but not always, forms a mass. When it does form a mass, it is usually called a "tumor." In brain tumors, uncontrolled cell division means that the size of the vegetation increases, and this quickly causes problems in confined spaces such as the intracranial cavity, as the mass encroaches on the brain's space, pushing it aside, leading to compression of brain tissue and increased intracranial pressure, as well as substantial destruction. According to the invention, "vesicle" or "tumor" also refers to a condition that would benefit from treatment with therapies targeting MUC17, particularly MUC17 expressed on the cell surface (such as MUC17-specific antibodies—including naked antibodies, antibody-drug conjugates (ADCs)). This condition includes chronic and acute disorders or diseases, including those pathological conditions that make mammals susceptible to the conditions (vesicles or tumors) discussed.

[0237] A growth or tumor can be benign, potentially malignant (precancerous), or malignant (cancerous). Malignant growths / tumors are often referred to as cancer. They typically invade and destroy surrounding tissues and can form metastases, meaning they spread to other parts of the body, tissues, or organs. A "primary tumor" is a tumor that grows at the anatomical site where tumor progression begins and continues to produce a cancerous mass. For example, gastrointestinal tumors occur when abnormal cells form in the gastrointestinal tract. Most cancers develop at their primary site but subsequently continue to form metastases or spread to other parts of the body (such as tissues and organs). These further tumors are secondary tumors. Most cancers continue to develop after their primary site, even after they have already spread to other parts of the body.

[0238] For the purposes of this invention, the terms “vesicle,” “tumor,” and “cancer” are used interchangeably, and they include primary tumors / cancers and secondary tumors / cancers (or “metastases”) as well as MRD. The term “minimal residual disease” (MRD) refers to evidence of the presence of a small number of residual cancer cells remaining in a patient’s body after cancer treatment, such as when the patient is in remission (the patient has no symptoms or signs of disease). Extremely small amounts of residual cancer cells are often undetectable by conventional means because standard tests used to assess or detect cancer are not sensitive enough to detect MRD. Today, molecular biological tests for MRD are highly sensitive, such as flow cytometry, PCR, and next-generation sequencing. These tests can measure the lowest levels of cancer cells in a tissue sample, sometimes as low as one cancer cell out of millions of normal cells. In the context of this invention, it is envisioned that the terms “prevention,” “treatment,” or “improvement” for vegetations also encompass “prevention, treatment, or improvement of MRD,” regardless of whether MRD is detected.

[0239] “Diseases associated with MUC17 or increased MUC17”, “MUC17-positive vegetations”, or “(MUC17-positive) vegetations” can be selected from the following group, which includes, but is not limited to…

[0240] "Diagnosis" or "medical diagnosis" is the process of determining which disorder or symptom can explain a subject's symptoms and signs. Typically, one or more diagnostic procedures, such as diagnostic tests or medical tests, are performed in this process. In medicine, the term "monitoring" refers to observing a disease, symptom, or one or more medical parameters over time. It can be performed by continuously measuring certain parameters using a medical monitor and / or by repeatedly performing medical tests. Diagnostic tests or medical tests are medical procedures performed to detect, diagnose, or monitor a disease, disease process, susceptibility, and / or determine the course of treatment. It is related to clinical chemistry and molecular diagnostics, and these procedures are typically performed in medical laboratories.

[0241] Medical therapy or treatment is an effort to cure or improve a disease. In the medical field, common treatments include pharmacological therapy. Pharmacological therapy (also known as drugs, pharmaceuticals, or medications) is used to diagnose, cure, treat, or prevent disease. Therefore, the term "treatment" refers to both therapeutic treatments and prophylactic or preventative measures.

[0242] In another aspect, the present invention also provides a method for detecting and / or quantifying MUC17 in a sample, the method comprising the following steps:

[0243] (a) Determining the MUC17 content in a sample using the antibody of the present invention or the detection system of the present invention; and

[0244] (b) Compare the MUC17 content determined in step (a) with the following:

[0245] (i) Predefined values ​​for MUC17 content,

[0246] (ii) The determined MUC17 content in the control sample, or

[0247] (c) The MUC17 content determined in samples obtained from the same source or subject at a previous time point.

[0248] For the purposes of this invention, the term "amount" or "content" of MUC17 may be used interchangeably with the terms "level," "quantity," or "concentration" of MUC17. A "predefined value for MUC17 content" may be a predetermined "cutoff value." This value may, for example, indicate that a certain level of MUC17 in a sample indicates a disease or MUC17-positive vegetation associated with MUC17 or an increased level of MUC17. For example, if the MUC17 content in a sample is determined to be three standard deviations above a predefined cutoff value, the subject from whom the sample was obtained is considered positive for gastrointestinal cancer (or other diseases as described herein). A "control sample" is typically obtained from a source having the same properties as the sample to be analyzed. A control sample may be a sample representing "normal" MUC17 content (e.g., representing a healthy subject) ("negative control sample") or a sample representing an "abnormally increased" level of MUC17 content ("positive control sample") (e.g., representing a subject with a disease as defined herein).

[0249] In another aspect, the present invention provides a method for diagnosing diseases associated with MUC17 or increased MUC17, the method comprising the following steps:

[0250] (a) Determining the MUC17 content in a sample using the antibody of the present invention or the detection system of the present invention; and

[0251] (b) Compare the MUC17 content determined in step (a) with the following:

[0252] (i) A predefined cutoff value for MUC17 levels indicating the absence of this disease, or

[0253] (ii) Represents the MUC17 content determined in control samples where this disease is absent.

[0254] A higher MUC17 content determined in step (a) compared to the predefined cutoff value in (i) or the MUC17 content determined in the control sample in (ii) indicates the presence of a disease associated with MUC17 or an increased MUC17.

[0255] In another aspect, the present invention also provides a method for monitoring the progression of diseases associated with MUC17 or increased MUC17, or for monitoring the response to treatment for diseases associated with MUC17 or increased MUC17, the method comprising the steps of:

[0256] (a) Using the antibody of the present invention or the detection system of the present invention, determine at a first time point the content of MUC17 in a biological sample obtained from a subject diagnosed with the disease;

[0257] (b) Using the antibody of the present invention, or using the detection system of the present invention, determining the MUC17 content in a biological sample obtained from the subject at a second (later) time point or after treatment; and

[0258] (c) Compare the MUC17 content determined in step (a) with the MUC17 content determined in step (b);

[0259] A higher MUC17 content determined in step (a) compared to the MUC17 content determined in step (b) indicates that the disease is progressing, and / or a lower MUC17 content determined in step (a) compared to the MUC17 content determined in step (b) indicates that the disease is entering a remission phase or that the disease is responding to the treatment.

[0260] For the methods described above, the “sample” is envisioned to be a biological sample, such as a human biological sample. The sample may be a (human) tissue sample or a (human) cell culture. The “sample” may also be obtained from a human subject, preferably a subject suspected of having or diagnosed with a disease associated with MUC17 or increased MUC17, or a subject who has received treatment for a disease associated with MUC17 or increased MUC17, as defined above.

[0261] For the above method, it is envisioned that "diseases associated with MUC17 or increased MUC17" could be MUC17-positive vegetations. Such diseases or MUC17-positive vegetations could be in the following group: gastric cancer, esophageal cancer, gastroesophageal cancer (including gastroesophageal junction cancer), gastrointestinal cancer, and pancreatic cancer.

[0262] The present invention also considers the following items:

[0263] Project 1. A monoclonal antibody that may or may not bind to soluble MUC17 (MUC17), wherein the binding of the antibody to MUC17 occurs in the presence of a second monoclonal antibody that binds to MUC17.

[0264] Project 2. The monoclonal antibody according to Project 1, wherein MUC17 has the amino acid sequence as depicted in SEQ ID NO:1.

[0265] Item 3. The monoclonal antibody according to Item 1 or 2, wherein the binding of the monoclonal antibody to MUC17 occurs in the presence of a third antibody that binds to MUC17.

[0266] Item 4: A monoclonal antibody according to any one of the preceding items, wherein the monoclonal antibody comprises a rodent, such as a mouse or rabbit, VH region and / or VL region.

[0267] Item 5. The monoclonal antibody according to any one of the preceding items, wherein the affinity (KD) of the monoclonal antibody for MUC17 is about ≤10. -7 M, ≤10 -8 M, ≤10 -9 M, or ≤10 -10 M.

[0268] Project 6. The monoclonal antibody according to Project 5, wherein the affinity is determined in a Biacore assay.

[0269] Item 7. A monoclonal antibody as described in any of the preceding items, wherein the monoclonal antibody:

[0270] a) Contains a VH region, which includes VH-CDR1 as depicted in SEQ ID NO:2, VH-CDR2 as depicted in SEQ ID NO:3, and VH-CDR3 as depicted in SEQ ID NO:4; and a VL region, which includes VL-CDR1 as depicted in SEQ ID NO:6, VL-CDR2 as depicted in SEQ ID NO:7, and VL-CDR3 as depicted in SEQ ID NO:8;

[0271] b) The antibody binds to the same MUC17 epitope as the antibody in a) or competes with the antibody in a) for binding to MUC17;

[0272] c) The antibody binds to the same MUC17 epitope as b) or competes with the antibody in b) for binding to MUC17; or

[0273] d) The antibody binds to the same MUC17 epitope as c) or competes with the antibody in c) for binding to MUC17.

[0274] Item 8. The monoclonal antibody according to Item 7, wherein the monoclonal antibody comprises

[0275] a) A VH region comprising an amino acid sequence having at least 60%, 65%, or 70% homology to the VH region contained in SEQ ID NO:5, preferably at least 75% or 80% homology, more preferably at least 85%, 90%, 91%, 92%, 93%, or 94% homology, and most preferably 95%, 96%, 97%, 98%, or 99% homology; a VL region comprising an amino acid sequence having at least 60%, 65%, or 70% homology to the VL region contained in SEQ ID NO:9, preferably at least 75% or 80% homology, more preferably at least 85%, 90%, 91%, 92%, 93%, or 94% homology, and most preferably 95%, 96%, 97%, 98%, or 99% homology; and optionally comprising VH-CDR1 as depicted in SEQ ID NO:2, as depicted in SEQ ID NO:5. VH-CDR2 as depicted in NO:3 and VH-CDR3 as depicted in SEQ ID NO:4, and optionally including VL-CDR1 as depicted in SEQ ID NO:6, VL-CDR2 as depicted in SEQ ID NO:7, and VL-CDR3 as depicted in SEQ ID NO:8.

[0276] Item 9. The monoclonal antibody according to Item 7, wherein the monoclonal antibody:

[0277] a) Contains the VH region included in SEQ ID NO:5;

[0278] b) Contains the VL region contained in SEQ ID NO:9;

[0279] c) Includes the VH region contained in SEQ ID NO:5 and the VL region contained in SEQ ID NO:9;

[0280] d) The antibody binds to the same MUC17 epitope as c) or competes with the antibody in c) for binding to MUC17;

[0281] e) The antibody binds to the same MUC17 epitope as d) or competes with the antibody of d) for binding to MUC17; or

[0282] f) The antibody binds to the same MUC17 epitope as e) or competes with the antibody for binding to MUC17.

[0283] Item 10. A monoclonal antibody according to any one of Items 6 to 9, wherein the binding to the MUC17 epitope is determined by epitope mapping with chimeric or mutated MUC17 molecules, site-directed mutagenesis (e.g., alanine scanning), high-throughput shotgun mutagenesis epitope mapping, cross-linked mass spectrometry, X-ray cocrystal analysis, cryo-electron microscopy, and hydrogen-deuterium exchange.

[0284] Item 11. The monoclonal antibody as described in any one of Items 6 to 9, wherein competition for binding to MUC17 is determined in a competitive ELISA assay, in an Octet competitive assay, or in a competitive assay using avidin-conjugated microparticles.

[0285] Item 12. A monoclonal antibody according to any one of Items 6 to 9 or 11, wherein competition for binding to MUC17 is defined as competition occurring between two test antibodies at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0286] Item 13. The monoclonal antibody according to any one of the preceding items, wherein the monoclonal antibody is an IgG, IgD, IgE, IgM or IgA antibody, preferably an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody.

[0287] Item 14. The monoclonal antibody according to any one of the preceding items, wherein the monoclonal antibody and / or the second monoclonal antibody bind to MUC17 in a biological sample, the biological sample preferably being a human biological sample, such as a (human) serum sample, (human) plasma sample, (human) blood sample, (human) bone marrow sample, (human) tissue sample, or tissue sample derived from the stomach, gastrointestinal tract, esophagus, gastroesophageal junction including the gastroesophageal junction, and pancreas.

[0288] Item 15. A polynucleotide encoding a monoclonal antibody according to any one of the preceding items.

[0289] Item 16. A vector comprising the polynucleotide as described in Item 15.

[0290] Item 17. A host cell, which is transformed or transfected with a polynucleotide according to Item 15 or a vector according to Item 16.

[0291] Item 18. A process for producing a monoclonal antibody according to any one of Items 1 to 14, the process comprising culturing a host cell according to Item 17 under conditions allowing expression of the monoclonal antibody and recovering the resulting monoclonal antibody from the culture.

[0292] Item 19. A composition comprising a monoclonal antibody produced according to any one of Items 1 to 14 or according to the process described in Item 18.

[0293] Item 20. A detection system comprising

[0294] a) The first monoclonal antibody that binds to MUC17, and

[0295] b) The first monoclonal antibody that binds to MUC17.

[0296] The binding of the first monoclonal antibody to MUC17 occurs in the presence of the second monoclonal antibody that is bound to MUC17, and / or the binding of the second monoclonal antibody to MUC17 occurs in the presence of the first monoclonal antibody that is bound to MUC17.

[0297] Item 21. The detection system according to Item 20, wherein MUC17 has the amino acid sequence as depicted in SEQ ID NO:1.

[0298] Item 22. The detection system according to Item 20 or 21, wherein the binding of the first monoclonal antibody to MUC17 and the binding of the second monoclonal antibody to MUC17 occur in the presence of a third antibody that binds to MUC17.

[0299] Item 23. The detection system according to any one of Items 20 to 23, wherein the first monoclonal antibody and / or the second monoclonal antibody comprises a mouse VH region and / or a mouse VL region.

[0300] Item 24. The detection system according to any one of Items 20 to 23, wherein the affinity (KD) of the first monoclonal antibody and / or the second monoclonal antibody for MUC17 is about ≤10. -7 M, ≤10 -8 M, ≤10 -9 M, or ≤10 -10 M.

[0301] Item 25. The detection system according to any one of Items 20 to 24, wherein the first monoclonal antibody:

[0302] a) Containing a VH region, which includes VH-CDR1 as depicted in SEQ ID NO:2, VH-CDR2 as depicted in SEQ ID NO:3, and VH-CDR3 as depicted in SEQ ID NO:4; and a VL region, which includes VL-CDR1 as depicted in SEQ ID NO:6, VL-CDR2 as depicted in SEQ ID NO:7, and VL-CDR3 as depicted in SEQ ID NO:8; or

[0303] b) Binds to the same MUC17 epitope as the antibody in a) or b), or competes with the antibody in a) for binding to MUC17.

[0304] Item 26. The detection system according to any one of Items 20 to 26, wherein the first monoclonal antibody and / or the second monoclonal antibody is an IgG, IgD, IgE, IgM or IgA antibody, preferably an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody.

[0305] Item 27. The detection system according to any one of Items 20 to 26, wherein the first monoclonal antibody is used as a capture antibody and the second monoclonal antibody is used as a detection antibody, or wherein the first monoclonal antibody is used as a detection antibody and the second monoclonal antibody is used as a capture antibody.

[0306] Item 28. Use of the monoclonal antibody according to any one of Items 1 to 14 or the detection system according to any one of Items 20 to 27 in the following respects:

[0307] - Detect MUC17 in the sample;

[0308] -Quantitative analysis of MUC17 in the sample;

[0309] - Diagnose diseases associated with MUC17 or increased MUC17;

[0310] - Stratify patients diagnosed with diseases associated with MUC17 or increased MUC17;

[0311] - Monitor the progression of diseases associated with MUC17 or increased MUC17; or

[0312] - Monitor the response to treatment for diseases associated with MUC17 or increased MUC17.

[0313] Item 29. A method for detecting and / or quantifying MUC17 in a sample, the method comprising the following steps:

[0314] (a) Determine the content of MUC17 in a sample using a monoclonal antibody (or derivative) according to any one of items 1 to 14, or using a detection system according to any one of items 20 to 27; and

[0315] (b) Compare the MUC17 content determined in step (a) with the following:

[0316] (i) Predefined values ​​for MUC17 content,

[0317] (ii) The determined MUC17 content in the control sample, or

[0318] (iii) The MUC17 content determined in samples obtained from the same source or subject at a previous time point.

[0319] Item 30. A method for diagnosing diseases associated with or increased MUC17, the method comprising the steps of: (a) determining the content of MUC17 in a sample, preferably a tissue sample for IHC, using a monoclonal antibody according to any one of Items 1 to 14, or using a detection system according to any one of Items 20 to 27; and (b) comparing the MUC17 content determined in step (a) with:

[0320] (i) A predefined cutoff value for MUC17 levels indicating the absence of this disease, or

[0321] (ii) Represents the MUC17 content determined in control samples where this disease is absent.

[0322] A higher MUC17 content determined in step (a) compared to the predefined cutoff value in (i) or the MUC17 content determined in the control sample in (ii) indicates the presence of a disease associated with MUC17 or an increased MUC17.

[0323] Item 31. A method for monitoring the progression of a disease associated with MUC17 or increased MUC17 or for monitoring the response to treatment for a disease associated with MUC17 or increased MUC17, the method comprising the steps of:

[0324] (a) Using a monoclonal antibody according to any one of items 1 to 14, or using a detection system according to any one of items 20 to 28, determine at a first time point the content of MUC17 in a biological sample obtained from a subject diagnosed with the disease;

[0325] (b) Using a monoclonal antibody according to any one of items 1 to 14, or using a detection system according to any one of items 20 to 28, determine the MUC17 content in a biological sample obtained from the subject at a second time point or after treatment; and

[0326] (c) Compare the MUC17 content determined in step (a) with the MUC17 content determined in step (b);

[0327] A higher MUC17 content determined in step (a) compared to the MUC17 content determined in step (b) indicates that the disease is progressing, and / or a lower MUC17 content determined in step (a) compared to the MUC17 content determined in step (b) indicates that the disease is entering a remission phase or that the disease is responding to the treatment.

[0328] Item 32. The use according to Item 28 or the method according to any one of Items 29 to 31, wherein the sample is a biological sample, preferably a human biological sample, such as a serum sample, plasma sample, blood sample, bone marrow sample, tissue sample or supernatant obtained from a cell culture of bone marrow mononuclear cells or peripheral blood mononuclear cells, preferably a tissue sample.

[0329] Item 33. The use according to Item 28 or 32 or the method according to any one of Items 29 to 32, wherein the sample is obtained from a human subject, preferably a human subject suspected of having or having a disease associated with MUC17 or increased MUC17, or a subject who has received treatment for a disease associated with MUC17 or increased MUC17.

[0330] Item 34. The use according to any one of Items 29, 32 or 33 or the method according to any one of Items 29 to 33, wherein the disease is selected from the group consisting of: gastric cancer, esophageal cancer, gastroesophageal cancer including gastroesophageal junction cancer, gastrointestinal cancer, and pancreatic cancer.

[0331] Item 35. A method for treating a patient suffering from at least one type of cancer, preferably a patient, the cancer being selected from the group consisting of: esophageal cancer, gastric cancer, gastrointestinal cancer, gastroesophageal cancer including gastroesophageal junction cancer, and / or pancreatic cancer, wherein the patient has increased MUC17 expression on cells of cancer cells, particularly colorectal cancer, gastric cancer, pancreatic cancer, colonic adenocarcinoma, esophageal cancer, pancreatic adenocarcinoma, rectal adenocarcinoma, and gastric adenocarcinoma.

[0332] Item 36. The treatment method according to Item 35, wherein the increased expression is determined in the method according to any one of Items 29-31, or includes use or product related to a diagnostic antibody or composition or drug containing the antibody used according to any one of the above-mentioned items.

[0333] Item 37. The treatment method according to items 35 and 36, the method comprising administering a drug to the patient, wherein the drug is selected from the group consisting of: small molecules, peptide drugs, antibodies and their derivatives, toxins, radiotherapy, and surgery.

[0334] Item 38. A treatment method according to any one of Items 35 to 37, wherein the drug is an antibody construct comprising at least two domains, one of which selectively binds to MUC17.

[0335] Item 39. A treatment method according to any one of Items 35 to 38, wherein the drug is an antibody construct comprising at least two domains, wherein the other domain selectively binds to CD3, human CD3, particularly human CD3ε chain.

[0336] Item 40. A treatment method according to any one of Items 35 to 39, wherein the drug is an antibody construct further comprising a third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker.

[0337] Item 41. The treatment method according to any one of Items 35 to 40, wherein the drug is a single-chain antibody construct.

[0338] Item 42. A treatment method according to any one of Items 35 to 41, wherein the drug is an antibody construct, wherein the third domain comprises, in the order of amino to carboxyl: hinge-CH2-CH3-connector-hinge-CH2-CH3.

[0339] Item 43. The treatment method according to any one of Items 35 to 42, wherein the drug is an antibody construct, wherein each of the polypeptide monomers of the third domain has an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO:22-29.

[0340] Item 44. The treatment method according to any one of Items 35 to 43, wherein the drug is an antibody construct, wherein each of the polypeptide monomers has an amino acid sequence selected from SEQ ID NO:22-29.

[0341] Item 45. A treatment method according to any one of items 35 to 44, wherein the drug is an antibody construct, wherein the CH2 domain contains an intradomain cysteine ​​disulfide bridge.

[0342] Item 46. A treatment method according to any one of Items 35 to 45, wherein the drug is an antibody construct, wherein (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain.

[0343] Item 47. The treatment method according to any one of Items 35 to 46, wherein the drug is an antibody construct, wherein the first domain and the second domain are fused to the third domain via a peptide linker.

[0344] Item 48. A treatment method according to any one of Items 35 to 47, wherein the drug is an antibody construct, wherein the antibody construct comprises, in the order from amino to carboxyl: (a) the first domain; (b) a peptide linker, the peptide linker preferably having an amino acid sequence selected from the group consisting of SEQ ID NO:10-12; and (c) the second domain.

[0345] Item 49. A treatment method according to any one of Items 35 to 48, wherein the drug is an antibody construct, wherein the antibody construct further comprises, in the order from amino to carboxyl: (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 17, 18, 19 and 20; (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 NO: 13, 14, 15 and 16; and (g) a second polypeptide monomer of the third domain.

[0346] Item 50. The treatment method according to any one of Items 35 to 49, wherein the drug is an antibody construct, wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO. 526 (aa 4171 to 4296 according to uniprot Q685J3).

[0347] Item 51. The treatment method according to any one of Items 35 to 50, wherein the drug is an antibody construct, wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO. 527 (aa 4184 to 4291 according to uniprot Q685J3).

[0348] Item 52. The treatment method according to any one of Items 35 to 51, wherein the drug is an antibody construct, wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO. 528 (aa 4131 to 4243 according to uniprot Q685J3).

[0349] Item 53. The treatment method according to any one of Items 35 to 52, wherein the drug is an antibody construct, wherein a first domain of the antibody construct binds to an epitope within MUC17 corresponding to SEQ ID NO. 529 (aa 4244 to 4389 according to uniprot Q685J3).

[0350] Item 54. The treatment method according to any one of Items 35 to 53, wherein the drug is an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO. 528 (aa 4131 to 4243 according to uniprot Q685J3), but does not bind to an epitope in MUC17 corresponding to SEQ ID NO. 529 (aa 4244 to 4389 according to uniprot Q685J3).

[0351] Item 55. A treatment method according to any one of items 35 to 54, wherein the drug is an antibody construct, wherein a first domain of the antibody construct binds to an epitope in MUC17 corresponding to SEQ ID NO. 530 (aa 4171 to 4390 according to uniprot Q685J3) or SEQ ID NO. 531 (aa 4184 to 4390 according to uniprot Q685J3), but does not bind to an epitope in MUC17 corresponding to SEQ ID NO. 532 (aa 4291 to 4390 according to uniprot Q685J3) or an epitope in MUC17 corresponding to SEQ ID NO. 533 (aa 4341 to 4390 according to uniprot Q685J3).

[0352] Item 56. A treatment method according to any one of items 35 to 55, wherein the drug is an antibody construct, wherein the ratio between cytotoxicity and binding affinity (EC50 / KD)*1000 is less than 250, wherein the cytotoxicity is determined with NUGC-4 cells as target cells and huPBMCs as effector cells, and wherein the binding affinity is determined by a surface plasmon resonance-based assay.

[0353] Item 57. A treatment method according to any one of Items 35 to 56, wherein the drug is an antibody construct, wherein the ratio between cytotoxicity and binding affinity (EC50 / KD)*1000 is less than 125, wherein the cytotoxicity is determined with NUGC-4 cells as target cells and huPBMCs as effector cells, and wherein the binding affinity is determined by a surface plasmon resonance-based assay.

[0354] Item 58. A treatment method according to any one of Items 35 to 57, wherein the drug is an antibody construct, wherein the ratio between cytotoxicity and binding affinity (EC50 / KD)*1000 is less than 21, wherein the cytotoxicity is determined with NUGC-4 cells as target cells and huPBMCs as effector cells, and wherein the binding affinity is determined by a surface plasmon resonance-based assay.

[0355] Item 59. A treatment method according to any one of Items 35 to 58, wherein the drug is an antibody construct, wherein the first binding domain comprises a VH region containing a CDR-H1, CDR-H2, and CDR-H3 selected from: (a) CDR-H1 as depicted in SEQ ID NO. 31, CDR-H2 as depicted in SEQ ID NO. 32, and CDR-H3 as depicted in SEQ ID NO. 33; (b) CDR-H1 as depicted in SEQ ID NO. 42, CDR-H2 as depicted in SEQ ID NO. 43, and CDR-H3 as depicted in SEQ ID NO. 44; (c) CDR-H1 as depicted in SEQ ID NO. 53, CDR-H2 as depicted in SEQ ID NO. 54, and CDR-H3 as depicted in SEQ ID NO. 55; (d) CDR-H1 as depicted in SEQ ID NO. 64, and CDR-H3 as depicted in SEQ ID NO. 55. (e) CDR-H1 as depicted in SEQ ID NO. 65, and CDR-H3 as depicted in SEQ ID NO. 76; (f) CDR-H1 as depicted in SEQ ID NO. 86, CDR-H2 as depicted in SEQ ID NO. 87, and CDR-H3 as depicted in SEQ ID NO. 88; (g) CDR-H1 as depicted in SEQ ID NO. 97, CDR-H2 as depicted in SEQ ID NO. 98, and CDR-H3 as depicted in SEQ ID NO. 99; (h) CDR-H1 as depicted in SEQ ID NO. 108, CDR-H2 as depicted in SEQ ID NO. 109, and CDR-H3 as depicted in SEQ ID NO. 110; (i) as depicted in SEQ ID NO. 65; CDR-H1 as depicted in SEQ ID NO. 119, CDR-H2 as depicted in SEQ ID NO. 120, and CDR-H3 as depicted in SEQ ID NO. 121; (j) CDR-H1 as depicted in SEQ ID NO. 130, CDR-H2 as depicted in SEQ ID NO. 131, and CDR-H3 as depicted in SEQ ID NO. 132; (k) CDR-H1 as depicted in SEQ ID NO. 141, CDR-H2 as depicted in SEQ ID NO. 142, and CDR-H3 as depicted in SEQ ID NO. 143; (l) CDR-H1 as depicted in SEQ ID NO. 152, CDR-H2 as depicted in SEQ ID NO. 153, and CDR-H3 as depicted in SEQ ID NO. 119.CDR-H3 as depicted in SEQ ID NO. 154; (m) CDR-H1 as depicted in SEQ ID NO. 163, CDR-H2 as depicted in SEQ ID NO. 164, and CDR-H3 as depicted in SEQ ID NO. 165; (n) CDR-H1 as depicted in SEQ ID NO. 174, CDR-H2 as depicted in SEQ ID NO. 175, and CDR-H3 as depicted in SEQ ID NO. 176; (o) CDR-H1 as depicted in SEQ ID NO. 185, CDR-H2 as depicted in SEQ ID NO. 186, and CDR-H3 as depicted in SEQ ID NO. 187; (p) CDR-H1 as depicted in SEQ ID NO. 196, CDR-H2 as depicted in SEQ ID NO. 197, and CDR-H3 as depicted in SEQ ID NO. 198; (q) as depicted in SEQ ID NO. CDR-H1 as depicted in NO. 207, CDR-H2 as depicted in SEQ ID NO. 208, and CDR-H3 as depicted in SEQ ID NO. 209; (r) CDR-H1 as depicted in SEQ ID NO. 218, CDR-H2 as depicted in SEQ ID NO. 219, and CDR-H3 as depicted in SEQ ID NO. 220; (s) CDR-H1 as depicted in SEQ ID NO. 229, CDR-H2 as depicted in SEQ ID NO. 230, and CDR-H3 as depicted in SEQ ID NO. 231; (t) CDR-H1 as depicted in SEQ ID NO. 240, CDR-H2 as depicted in SEQ ID NO. 241, and CDR-H3 as depicted in SEQ ID NO. 242; (u) CDR-H1 as depicted in SEQ ID NO. 251, and CDR-H3 as depicted in SEQ ID NO. 207, CDR-H2 as depicted in SEQ ID NO. 208, and CDR-H3 as depicted in SEQ ID NO. 209; (r) CDR-H1 as depicted in SEQ ID NO. 218, CDR-H2 as depicted in SEQ ID NO. 219, and CDR-H3 as depicted in SEQ ID NO. 220; (s) CDR-H1 as depicted in SEQ ID NO. 229, CDR-H2 as depicted in SEQ ID NO. 230, and CDR-H3 as depicted in SEQ ID NO. 231; (t) CDR-H1 as depicted in SEQ ID NO. 240, CDR-H2 as depicted in SEQ ID NO. 241, and CDR-H3 as depicted in SEQ ID NO. 242; (u) CDR-H1 as (v) CDR-H2 as depicted in SEQ ID NO. 252, and CDR-H3 as depicted in SEQ ID NO. 253; (v) CDR-H1 as depicted in SEQ ID NO. 262, CDR-H2 as depicted in SEQ ID NO. 263, and CDR-H3 as depicted in SEQ ID NO. 264; (w) CDR-H1 as depicted in SEQ ID NO. 273, CDR-H2 as depicted in SEQ ID NO. 274, and CDR-H3 as depicted in SEQ ID NO. 275; (x) CDR-H1 as depicted in SEQ ID NO. 284, CDR-H2 as depicted in SEQ ID NO. 285, and CDR-H3 as depicted in SEQ ID NO. 286; (y) CDR-H1 as depicted in SEQ ID NO. 295, and CDR-H3 as depicted in SEQ ID NO. 253.CDR-H2 as depicted in SEQ ID NO. 296, and CDR-H3 as depicted in SEQ ID NO. 297; (z) CDR-H1 as depicted in SEQ ID NO. 306, CDR-H2 as depicted in SEQ ID NO. 307, and CDR-H3 as depicted in SEQ ID NO. 308; (aa) CDR-H1 as depicted in SEQ ID NO. 317, CDR-H2 as depicted in SEQ ID NO. 318, and CDR-H3 as depicted in SEQ ID NO. 319; (ab) CDR-H1 as depicted in SEQ ID NO. 328, CDR-H2 as depicted in SEQ ID NO. 329, and CDR-H3 as depicted in SEQ ID NO. 330; (ac) CDR-H1 as depicted in SEQ ID NO. 339, CDR-H2 as depicted in SEQ ID NO. 340, and CDR-H3 as depicted in SEQ ID NO. 296, and CDR-H3 as depicted in SEQ ID NO. 307; CDR-H3 as depicted in NO. 341; (ad) CDR-H1 as depicted in SEQ ID NO. 350, CDR-H2 as depicted in SEQ ID NO. 351, and CDR-H3 as depicted in SEQ ID NO. 352; (ae) CDR-H1 as depicted in SEQ ID NO. 361, CDR-H2 as depicted in SEQ ID NO. 362, and CDR-H3 as depicted in SEQ ID NO. 363; (af) CDR-H1 as depicted in SEQ ID NO. 372, CDR-H2 as depicted in SEQ ID NO. 373, and CDR-H3 as depicted in SEQ ID NO. 374; (ag) CDR-H1 as depicted in SEQ ID NO. 383, CDR-H2 as depicted in SEQ ID NO. 384, and CDR-H3 as depicted in SEQ ID NO. 385; (ah) as depicted in SEQ ID NO. CDR-H1 as depicted in NO. 394, CDR-H2 as depicted in SEQ ID NO. 395, and CDR-H3 as depicted in SEQ ID NO. 396; (ai) CDR-H1 as depicted in SEQ ID NO. 405, CDR-H2 as depicted in SEQ ID NO. 406, and CDR-H3 as depicted in SEQ ID NO. 407; (aj) CDR-H1 as depicted in SEQ ID NO. 416, CDR-H2 as depicted in SEQ ID NO. 417, and CDR-H3 as depicted in SEQ ID NO. 418; (ak) CDR-H1 as depicted in SEQ ID NO. 427, CDR-H2 as depicted in SEQ ID NO. 428, and CDR-H3 as depicted in SEQ ID NO. 394.CDR-H3 as depicted in SEQ ID NO. 429; (al) CDR-H1 as depicted in SEQ ID NO. 438, CDR-H2 as depicted in SEQ ID NO. 439, and CDR-H3 as depicted in SEQ ID NO. 440; (am) CDR-H1 as depicted in SEQ ID NO. 449, CDR-H2 as depicted in SEQ ID NO. 450, and CDR-H3 as depicted in SEQ ID NO. 451; (an) CDR-H1 as depicted in SEQ ID NO. 460, CDR-H2 as depicted in SEQ ID NO. 461, and CDR-H3 as depicted in SEQ ID NO. 462; (ao) CDR-H1 as depicted in SEQ ID NO. 471, CDR-H2 as depicted in SEQ ID NO. 472, and CDR-H3 as depicted in SEQ ID NO. 473; (ap) as depicted in SEQ ID NO. The following are examples of CDR-H1 as depicted in NO. 482, CDR-H2 as depicted in SEQ ID NO. 483, and CDR-H3 as depicted in SEQ ID NO. 484; (aq) the following are examples of CDR-H1 as depicted in SEQ ID NO. 493, CDR-H2 as depicted in SEQ ID NO. 494, and CDR-H3 as depicted in SEQ ID NO. 495; (ar) the following are examples of CDR-H1 as depicted in SEQ ID NO. 504, CDR-H2 as depicted in SEQ ID NO. 505, and CDR-H3 as depicted in SEQ ID NO. 506; and (as) the following are examples of CDR-H1 as depicted in SEQ ID NO. 515, CDR-H2 as depicted in SEQ ID NO. 516, and CDR-H3 as depicted in SEQ ID NO. 517; wherein preferably is (c) the following are examples of CDR-H1 as depicted in SEQ ID NO. 53, CDR-H2 as depicted in SEQ ID NO. 482, CDR-H2 as depicted in SEQ ID NO. 483, CDR-H3 as depicted in SEQ ID NO. 484; (aq) the following are examples of CDR-H1 as depicted in SEQ ID NO. 493, CDR-H2 as depicted in SEQ ID NO. 494, and CDR-H3 as depicted in SEQ ID NO. 495; (ar) the following are examples of CDR-H1 as depicted in SEQ ID NO. 504, CDR-H2 as depicted in SEQ ID NO. 505, and CDR-H3 as depicted in SEQ ID NO. 506; and (as) the following are examples of CDR-H1 as depicted in SEQ ID NO. 515, CDR-H CDR-H2 as depicted in SEQ ID NO. 54, and CDR-H3 as depicted in SEQ ID NO. 55; (n) CDR-H1 as depicted in SEQ ID NO. 174, CDR-H2 as depicted in SEQ ID NO. 175, and CDR-H3 as depicted in SEQ ID NO. 176; (ac) CDR-H1 as depicted in SEQ ID NO. 339, CDR-H2 as depicted in SEQ ID NO. 340, and CDR-H3 as depicted in SEQ ID NO. 341; and (aj) CDR-H1 as depicted in SEQ ID NO. 416, CDR-H2 as depicted in SEQ ID NO. 417, and CDR-H3 as depicted in SEQ ID NO. 418.

[0356] 60. The treatment method according to any one of items 35 to 59, wherein the drug is an antibody construct, wherein the first binding domain comprises a VL region containing a CDR-H1, CDR-L2, and CDR-L3 selected from: (a) CDR-L1 as depicted in SEQ ID NO. 34, CDR-L2 as depicted in SEQ ID NO. 35, and CDR-L3 as depicted in SEQ ID NO. 36; (b) CDR-L1 as depicted in SEQ ID NO. 45, CDR-L2 as depicted in SEQ ID NO. 46, and CDR-L3 as depicted in SEQ ID NO. 47; (c) CDR-L1 as depicted in SEQ ID NO. 56, CDR-L2 as depicted in SEQ ID NO. 57, and CDR-L3 as depicted in SEQ ID NO. 58; (d) CDR-L1 as depicted in SEQ ID NO. 67, and CDR-L3 as depicted in SEQ ID NO. 59. (e) CDR-L1 as depicted in SEQ ID NO. 68, and CDR-L3 as depicted in SEQ ID NO. 79; (f) CDR-L1 as depicted in SEQ ID NO. 78, CDR-L2 as depicted in SEQ ID NO. 79, and CDR-L3 as depicted in SEQ ID NO. 80; (g) CDR-L1 as depicted in SEQ ID NO. 89, CDR-L2 as depicted in SEQ ID NO. 90, and CDR-L3 as depicted in SEQ ID NO. 91; (h) CDR-L1 as depicted in SEQ ID NO. 111, CDR-L2 as depicted in SEQ ID NO. 112, and CDR-L3 as depicted in SEQ ID NO. 69; The following are examples of CDR-L3 as depicted in NO. 113: (i) CDR-L1 as depicted in SEQ ID NO. 122, CDR-L2 as depicted in SEQ ID NO. 123, and CDR-L3 as depicted in SEQ ID NO. 124; (j) CDR-L1 as depicted in SEQ ID NO. 133, CDR-L2 as depicted in SEQ ID NO. 134, and CDR-L3 as depicted in SEQ ID NO. 135; (k) CDR-L1 as depicted in SEQ ID NO. 144, CDR-L2 as depicted in SEQ ID NO. 145, and CDR-L3 as depicted in SEQ ID NO. 146; (l) CDR-L1 as depicted in SEQ ID NO. 155, CDR-L2 as depicted in SEQ ID NO. 156, and CDR-L3 as depicted in SEQ ID NO. 113.CDR-L3 as depicted in SEQ ID NO. 157; (m) CDR-L1 as depicted in SEQ ID NO. 166, CDR-L2 as depicted in SEQ ID NO. 167, and CDR-L3 as depicted in SEQ ID NO. 168; (n) CDR-L1 as depicted in SEQ ID NO. 177, CDR-L2 as depicted in SEQ ID NO. 178, and CDR-L3 as depicted in SEQ ID NO. 179; (o) CDR-L1 as depicted in SEQ ID NO. 188, CDR-L2 as depicted in SEQ ID NO. 189, and CDR-L3 as depicted in SEQ ID NO. 190; (p) CDR-L1 as depicted in SEQ ID NO. 199, CDR-L2 as depicted in SEQ ID NO. 200, and CDR-L3 as depicted in SEQ ID NO. 201; (q) as depicted in SEQ ID NO. CDR-L1 as depicted in NO. 210, CDR-L2 as depicted in SEQ ID NO. 211, and CDR-L3 as depicted in SEQ ID NO. 212; (r) CDR-L1 as depicted in SEQ ID NO. 221, CDR-L2 as depicted in SEQ ID NO. 222, and CDR-L3 as depicted in SEQ ID NO. 223; (s) CDR-L1 as depicted in SEQ ID NO. 232, CDR-L2 as depicted in SEQ ID NO. 233, and CDR-L3 as depicted in SEQ ID NO. 234; (t) CDR-L1 as depicted in SEQ ID NO. 243, CDR-L2 as depicted in SEQ ID NO. 244, and CDR-L3 as depicted in SEQ ID NO. 245; (u) CDR-L1 as depicted in SEQ ID NO. 254, and CDR-L3 as depicted in SEQ ID NO. 210, CDR-L2 as depicted in SEQ ID NO. 211, and CDR-L3 as depicted in SEQ ID NO. 212; (r) CDR-L1 as depicted in SEQ ID NO. 221, CDR-L2 as depicted in SEQ ID NO. 222, and CDR-L3 as depicted in SEQ ID NO. 223; (s) CDR-L1 as depicted in SEQ ID NO. 232, CDR-L2 as depicted in SEQ ID NO. 233, and CDR-L3 as depicted in SEQ ID NO. 234; (t) CDR-L1 as depicted in SEQ ID NO. 243, CDR-L2 as depicted in SEQ ID NO. 244, and CDR-L3 as depicted in SEQ ID NO. 245; (u) CDR-L1 as (v) CDR-L2 as depicted in SEQ ID NO. 255, and CDR-L3 as depicted in SEQ ID NO. 256; (v) CDR-L1 as depicted in SEQ ID NO. 265, CDR-L2 as depicted in SEQ ID NO. 266, and CDR-L3 as depicted in SEQ ID NO. 267; (w) CDR-L1 as depicted in SEQ ID NO. 276, CDR-L2 as depicted in SEQ ID NO. 277, and CDR-L3 as depicted in SEQ ID NO. 278; (x) CDR-L1 as depicted in SEQ ID NO. 287, CDR-L2 as depicted in SEQ ID NO. 288, and CDR-L3 as depicted in SEQ ID NO. 289; (y) CDR-L1 as depicted in SEQ ID NO. 298, and CDR-L3 as depicted in SEQ ID NO. 255.(z) CDR-L2 as depicted in SEQ ID NO. 309, and CDR-L3 as depicted in SEQ ID NO. 310; and CDR-L3 as depicted in SEQ ID NO. 311; (aa) CDR-L1 as depicted in SEQ ID NO. 320, CDR-L2 as depicted in SEQ ID NO. 321, and CDR-L3 as depicted in SEQ ID NO. 322; (ab) CDR-L1 as depicted in SEQ ID NO. 331, CDR-L2 as depicted in SEQ ID NO. 332, and CDR-L3 as depicted in SEQ ID NO. 333; (ac) CDR-L1 as depicted in SEQ ID NO. 342, CDR-L2 as depicted in SEQ ID NO. 343, and CDR-L3 as depicted in SEQ ID NO. 309; CDR-L3 as depicted in NO. 344; (ad) CDR-L1 as depicted in SEQ ID NO. 353, CDR-L2 as depicted in SEQ ID NO. 354, and CDR-L3 as depicted in SEQ ID NO. 355; (ae) CDR-L1 as depicted in SEQ ID NO. 364, CDR-L2 as depicted in SEQ ID NO. 365, and CDR-L3 as depicted in SEQ ID NO. 366; (af) CDR-L1 as depicted in SEQ ID NO. 375, CDR-L2 as depicted in SEQ ID NO. 376, and CDR-L3 as depicted in SEQ ID NO. 377; (ag) CDR-L1 as depicted in SEQ ID NO. 386, CDR-L2 as depicted in SEQ ID NO. 387, and CDR-L3 as depicted in SEQ ID NO. 388; (ah) as depicted in SEQ ID NO. CDR-L1 as depicted in NO. 397, CDR-L2 as depicted in SEQ ID NO. 398, and CDR-L3 as depicted in SEQ ID NO. 399; (ai) CDR-L1 as depicted in SEQ ID NO. 408, CDR-L2 as depicted in SEQ ID NO. 409, and CDR-L3 as depicted in SEQ ID NO. 410; (aj) CDR-L1 as depicted in SEQ ID NO. 419, CDR-L2 as depicted in SEQ ID NO. 420, and CDR-L3 as depicted in SEQ ID NO. 421; (ak) CDR-L1 as depicted in SEQ ID NO. 430, CDR-L2 as depicted in SEQ ID NO. 431, and CDR-L3 as depicted in SEQ ID NO. 397.The CDR-L3 depicted in SEQ ID NO. 432; (al) the CDR-L1 depicted in SEQ ID NO. 441, the CDR-L2 depicted in SEQ ID NO. 442, and the CDR-L3 depicted in SEQ ID NO. 443; (am) the CDR-L1 depicted in SEQ ID NO. 452, the CDR-L2 depicted in SEQ ID NO. 453, and the CDR-L3 depicted in SEQ ID NO. 454; (an) the CDR-L1 depicted in SEQ ID NO. 463, the CDR-L2 depicted in SEQ ID NO. 464, and the CDR-L3 depicted in SEQ ID NO. 465; (ao) the CDR-L1 depicted in SEQ ID NO. 474, the CDR-L2 depicted in SEQ ID NO. 475, and the CDR-L3 depicted in SEQ ID NO. 476; (ap) the CDR-L3 depicted in SEQ ID NO. 432; (a) the CDR-L1 depicted in SEQ ID NO. 441, the CDR-L2 depicted in SEQ ID NO. 442, and the CDR-L3 depicted in SEQ ID NO. 443; (a) the CDR-L1 depicted in SEQ ID NO. 452, the CDR-L2 depicted in SEQ ID NO. 453, and the CDR-L3 depicted in SEQ ID NO. 454 ...3 depicted in SEQ ID NO. 453, the CDR-L2 depicted in SEQ ID NO. 454, and the The following are examples of CDR-L1 as depicted in NO. 485, CDR-L2 as depicted in SEQ ID NO. 486, and CDR-L3 as depicted in SEQ ID NO. 487; (aq) the following are examples of CDR-L1 as depicted in SEQ ID NO. 496, CDR-L2 as depicted in SEQ ID NO. 497, and CDR-L3 as depicted in SEQ ID NO. 498; (ar) the following are examples of CDR-L1 as depicted in SEQ ID NO. 507, CDR-L2 as depicted in SEQ ID NO. 508, and CDR-L3 as depicted in SEQ ID NO. 509; and (as) the following are examples of CDR-L1 as depicted in SEQ ID NO. 518, CDR-L2 as depicted in SEQ ID NO. 519, and CDR-L3 as depicted in SEQ ID NO. 520; wherein preferably is (c) the following are examples of CDR-L1 as depicted in SEQ ID NO. 56, CDR-L2 as depicted in SEQ ID NO. 485, CDR-L2 as depicted in SEQ ID NO. 486, CDR-L3 as depicted in SEQ ID NO. 487; (aq) the following are examples of CDR-L1 as depicted in SEQ ID NO. 496, CDR-L2 as depicted in SEQ ID NO. 497, and CDR-L3 as depicted in SEQ ID NO. 498; (ar) the following are examples of CDR-L1 as depicted in SEQ ID NO. 507, CDR-L2 as depicted in SEQ ID NO. 508, and CDR-L3 as depicted in SEQ ID NO. 509; and (as) the following are examples of CDR-L1 as depicted in SEQ ID NO. 518, CDR-L CDR-L2 as depicted in NO. 57, and CDR-L3 as depicted in SEQ ID NO. 58; (n) CDR-L1 as depicted in SEQ ID NO. 177, CDR-L2 as depicted in SEQ ID NO. 178, and CDR-L3 as depicted in SEQ ID NO. 179; (ac) CDR-L1 as depicted in SEQ ID NO. 342, CDR-L2 as depicted in SEQ ID NO. 343, and CDR-L3 as depicted in SEQ ID NO. 344; and (aj) CDR-L1 as depicted in SEQ ID NO. 419, CDR-L2 as depicted in SEQ ID NO. 420, and CDR-L3 as depicted in SEQ ID NO. 421.

[0357] Item 61. A treatment method according to any one of Items 35 to 60, wherein the drug is an antibody construct, wherein the first binding domain comprises a VL region and a VH region selected from the group consisting of: (a) a VL region as depicted in SEQ ID NO. 38 and a VH region as depicted in SEQ ID NO. 37; (b) a VL region as depicted in SEQ ID NO. 49 and a VH region as depicted in SEQ ID NO. 48; (c) a VL region as depicted in SEQ ID NO. 60 and a VH region as depicted in SEQ ID NO. 59; (d) a VL region as depicted in SEQ ID NO. 71 and a VH region as depicted in SEQ ID NO. 70; (e) a VL region as depicted in SEQ ID NO. 82 and a VH region as depicted in SEQ ID NO. 81; (f) a VL region as depicted in SEQ ID NO. 93 and a VH region as depicted in SEQ ID NO. 92; (g) a VL region as depicted in SEQ ID NO. 93 and a VH region as depicted in SEQ ID NO. 92; (g) a VL region as depicted in SEQ ID NO. 93 and a VH region as depicted in SEQ ID NO. 94; (v) a VL region as depicted in SEQ ID NO. 95; (v) a VL region as depicted in SEQ ID NO. 96; (v) a VH region as depicted in SEQ ID NO. 97; (v) a VL region as depicted in SEQ ID NO. 98; (v) a VH region as depicted in SEQ ID NO. 99; (v) a VL region as depicted in SEQ ID NO. 99; (v) a VH region as depicted in SEQ ID NO. 99; (v) a VL region as depicted in SEQ ID NO. 99; (h) The VL region depicted in SEQ ID NO. 104 and the VH region depicted in SEQ ID NO. 103; (i) The VL region depicted in SEQ ID NO. 115 and the VH region depicted in SEQ ID NO. 114; (j) The VL region depicted in SEQ ID NO. 126 and the VH region depicted in SEQ ID NO. 125; (k) The VL region depicted in SEQ ID NO. 137 and the VH region depicted in SEQ ID NO. 136; (l) The VL region depicted in SEQ ID NO. 148 and the VH region depicted in SEQ ID NO. 147; (m) The VL region depicted in SEQ ID NO. 170 and the VH region depicted in SEQ ID NO. 169; (n) The VL region depicted in SEQ ID NO. 181 and the VH region depicted in SEQ ID NO. 103; The VH region depicted in NO. 180; (o) the VL region depicted in SEQ ID NO. 192 and the VH region depicted in SEQ ID NO. 191; (p) the VL region depicted in SEQ ID NO. 203 and the VH region depicted in SEQ ID NO. 202; (q) the VL region depicted in SEQ ID NO. 214 and the VH region depicted in SEQ ID NO. 213; (r) the VL region depicted in SEQ ID NO. 226 and the VH region depicted in SEQ ID NO. 225; (s) the VL region depicted in SEQ ID NO. 236 and the VH region depicted in SEQ ID NO. 235; (t) the VL region depicted in SEQ ID NO. 247 and the VH region depicted in SEQ ID NO. 180.The VH region depicted in SEQ ID NO. 246; (u) the VL region depicted in SEQ ID NO. 258 and the VH region depicted in SEQ ID NO. 257; (v) the VL region depicted in SEQ ID NO. 269 and the VH region depicted in SEQ ID NO. 268; (w) the VL region depicted in SEQ ID NO. 280 and the VH region depicted in SEQ ID NO. 279; (x) the VL region depicted in SEQ ID NO. 292 and the VH region depicted in SEQ ID NO. 290; (y) the VL region depicted in SEQ ID NO. 302 and the VH region depicted in SEQ ID NO. 301; (z) the VL region depicted in SEQ ID NO. 313 and the VH region depicted in SEQ ID NO. 312; (aa) the VL region depicted in SEQ ID NO. 324 and the VH region depicted in SEQ ID NO. 246. The VH region depicted in NO. 323; (ab) the VL region depicted in SEQ ID NO. 335 and the VH region depicted in SEQ ID NO. 334; (ac) the VL region depicted in SEQ ID NO. 346 and the VH region depicted in SEQ ID NO. 345; (ad) the VL region depicted in SEQ ID NO. 357 and the VH region depicted in SEQ ID NO. 356; (ae) the VL region depicted in SEQ ID NO. 368 and the VH region depicted in SEQ ID NO. 367; (af) the VL region depicted in SEQ ID NO. 379 and the VH region depicted in SEQ ID NO. 378; (ag) the VL region depicted in SEQ ID NO. 390 and the VH region depicted in SEQ ID NO. 389; (ah) the VL region depicted in SEQ ID NO. 401 and the VH region depicted in SEQ ID NO. 334; The VH region depicted in NO. 400; (ai) the VL region depicted in SEQ ID NO. 412 and the VH region depicted in SEQ ID NO. 411; (aj) the VL region depicted in SEQ ID NO. 423 and the VH region depicted in SEQ ID NO. 422; (ak) the VL region depicted in SEQ ID NO. 434 and the VH region depicted in SEQ ID NO. 433; (al) the VL region depicted in SEQ ID NO. 445 and the VH region depicted in SEQ ID NO. 444; (am) the VL region depicted in SEQ ID NO. 456 and the VH region depicted in SEQ ID NO. 455; (an) the VL region depicted in SEQ ID NO. 467 and the VH region depicted in SEQ ID NO. 466; (ao) the VL region depicted in SEQ ID NO. 478 and the VH region depicted in SEQ ID NO. 411.The VH region depicted in SEQ ID NO. 477; (ap) the VL region depicted in SEQ ID NO. 489 and the VH region depicted in SEQ ID NO. 488; (aq) the VL region depicted in SEQ ID NO. 500 and the VH region depicted in SEQ ID NO. 499; (ar) the VL region depicted in SEQ ID NO. 511 and the VH region depicted in SEQ ID NO. 510; and (as) the VL region depicted in SEQ ID NO. 522 and the VH region depicted in SEQ ID NO. 521.

[0358] Item 62. The treatment method according to any one of Items 35 to 61, wherein the drug is an antibody construct comprising a sequence of amino acid sequences selected from any one of the following: SEQ ID NO: 39, 50, 61, 72, 83, 94, 105, 116, 127, 138, 149, 160, 171, 182, 193, 204, 215, 226, 237, 248, 259, 270, 281, 292, 303, 314, 325, 336, 347, 358, 369, 380, 391, 402, 413, 424, 435, 446, 457, 468, 479, 490, 501, 512, and 523.

[0359] Item 63. A treatment method according to any one of Items 35 to 62, wherein the drug is an antibody construct, wherein the antibody construct comprises, in the order amino to carboxyl: (a) the first domain having an amino acid sequence selected from the group consisting of: SEQ ID NO:39, 50, 61, 72, 83, 94, 105, 116, 127, 138, 149, 160, 171, 182, 193, 204, 215, 226, 237, 248, 259, 270, 281, 292, 303, 314, 325, 336, 347, 358, 369, 380, 391, 402, 413, 424, 435, 446, 457, 468, 479, 490, 501, 512, and 523; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO:10-12; (c) the second domain having an amino acid sequence selected from the group consisting of SEQ ID NO:10-12 of WO 2008 / 119567. NO:23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187, or the amino acid sequence as depicted in SEQ ID NO:13.

[0360] Item 64. A treatment method according to any one of Items 35 to 63, wherein the medicament is an antibody construct, wherein the antibody construct further comprises, in the order amino to carboxyl: (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 17, 18, 19 and 20; (e) a first polypeptide monomer of the third domain having a polypeptide sequence selected from the group consisting of SEQ ID NO: 22-29; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16; and (g) a second polypeptide monomer of the third domain having a polypeptide sequence selected from the group consisting of SEQ ID NO: 22-29.

[0361] Item 65. A treatment method according to any one of Items 35 to 64, wherein the drug is an antibody construct having an amino acid sequence selected from the group consisting of: SEQ ID NO: 40, 41, 51, 52, 62, 63, 73, 74, 84, 85, 95, 96, 106, 107, 117, 118, 128, 129, 139, 140, 150, 151, 161, 162, 172, 173, 183, 184, 194, 195, 205, 206, 216, 217, 227, 228, 238, 239, 249, 250, 260, 261, 271, 272, 282, 283, 293, 294, 304, 305, 315, 316, 326, 32 7, 337, 338, 348, 349, 359, 360, 370, 371, 381, 382, ​​392, 393, 403, 404, 414, 415, 424, 426, 436, 437, 447, 448, 458, 459, 469, 470, 480, 481, 491, 492, 502, 503, 513, 514, 524, and 525, or having amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the said sequence.

[0362] The present invention further relates to the treatment methods listed below.

[0363] In a first aspect of the treatment method according to the invention, it is envisioned to administer an antibody construct comprising a first domain and a second domain, the first domain binding to MUC17 and the second domain binding to an extracellular surface site of the CD3ε chain of humans and macaques. In this aspect, it is further envisioned that the antibody construct comprises a third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain, and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker. In this aspect, it is further envisioned that the antibody construct is a single-chain antibody construct. In this aspect, it is further envisioned that the antibody construct has a third domain comprising, in the amino-to-carboxyl sequence: hinge-CH2-CH3-linker-hinge-CH2-CH3. In this aspect, it is further envisioned that each of the polypeptide monomers has an amino acid sequence having at least 90% identity with a sequence as defined in the sequence listing. In this aspect, it is further envisioned that the antibody construct is administered, wherein the CH2 domain comprises an intradomain cysteine ​​disulfide bridge.

[0364] Within these aspects, it is also contemplated to administer antibody constructs wherein (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain. Within these aspects, it is also contemplated to administer antibody constructs wherein the first and second domains are fused to the third domain via peptide linkers. In this regard, antibody constructs as disclosed in WO 2019 / 133961 are particularly considered, which is incorporated herein by reference. Antibody constructs claimed in the disclosure may be used in the treatment methods described herein. In the context of this invention, it is further contemplated to administer pharmaceutical compositions comprising antibody constructs as defined above.

[0365] The treatment methods according to the invention include the prevention, relief, prevention of its deterioration or recurrence, or improvement of diseases selected from: proliferative diseases, neoplastic diseases, cancers such as gastrointestinal cancers (such as gastric cancer, esophageal cancer, gastroesophageal (junction) cancer, or colorectal cancer) or pancreatic cancer, comprising the step of administering an antibody construct targeting MUC17 and CD3 to a subject in need. In the context of the invention, the administration of an antibody construct specifically targeting MUC17, which is associated with a diagnosis of malignancy, is provided. For this purpose, MUC17 is first identified as a gene upregulated relative to normal tissue expression in gastric tumors. In this regard, the inventive immunohistochemical methods described herein show that the MUC17 protein is expressed in 40%–77% of gastric tumors. Flow cytometry has also demonstrated that, with the exception of some pancreatic cancer cell lines and colorectal cancer cell lines, the MUC17 protein is expressed on the cell surface of gastric cancer cell lines and esophageal cancer cell lines. It has even been shown that this expression is particularly high in gastric tumors in Chinese patients. Therefore, MUC17 was identified as a potent target associated with gastrointestinal cancers, namely gastric cancer, small bowel cancer, colorectal cancer (colon cancer), esophageal cancer, and pancreatic cancer. In the context of this invention, the administered antibody construct exhibits binding affinity, potent cytotoxic activity, and is the most stable spectrum for the SEA domain. The administered antibody construct may have cysteine ​​clamps, i.e., intramolecular disulfide bonds, in the target conjugate to enhance stability, and may also be in the form of a single-chain Fc (scFc) targeting MUC17 as a half-life extension (HLE) portion. Furthermore, the scFc, i.e., HLE, antibody construct can be administered intravenously by administration only once a week, once every two weeks, once every three weeks, or even once every four weeks or less. Therefore, the antibody construct used in the method of the present invention comprises a first domain that binds to MUC17; a second domain that binds to an extracellular domain of the CD3ε chain of humans and macaques; and a third domain optionally comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain, and a CH3 domain, wherein the two polypeptide monomers are fused to each other via peptide linkers. In one embodiment, the applied antibody construct comprises all three such domains.

[0366] The term "antibody construct" refers to a molecule whose structure and / or function are based on that of an antibody, such as a full-length or whole immunoglobulin molecule. Therefore, an antibody construct can bind to its specific target or antigen and / or be derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or fragment thereof. Furthermore, the domain that binds to its binding partner is understood herein as the binding domain of the antibody construct. Typically, the binding domain according to the invention contains minimum structural requirements for the antibody to allow target binding. These minimum requirements can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region), preferably all six CDRs. Alternative methods for defining minimum structural requirements for an antibody include defining the antibody epitope within the specific target structure, the protein domain of the target protein constituting the epitope region (epitope cluster), or by referring to a specific antibody competing with the epitope of the defined antibody. The antibodies upon which the constructs according to the invention are based include, for example, monoclonal, recombinant, chimeric, deimmunized, humanized, and human antibodies. The binding domains of the antibody constructs applied according to the invention may, for example, include the CDR group mentioned above. Preferably, those CDRs are contained within the framework of the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH); however, it does not necessarily include both. For example, the Fd fragment has two VH regions and typically retains some antigen-binding function of the intact antigen-binding domain. Other examples of antibody fragments, antibody variants, or binding domains include (1) Fab fragments, a monovalent fragment having VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, a bivalent fragment having two Fab fragments connected at the hinge region by disulfide bridges; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains on a single arm of the antibody; (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) separate complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), the latter being preferred (e.g., derived from scFV libraries). Examples of embodiments of the antibody constructs according to the present invention are described, for example, in the following: WO00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO2014 / 151910 and WO 2015 / 048272.

[0367] Additionally, within the definition of "binding domain" or "bound domain" are fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "r IgG" ("half antibody"). Antibody constructs applied according to the invention may also contain modified fragments of the antibody (also called antibody variants), such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, biantibodies, single-chain biantibodies, tandem biantibodies (Tandab), tandem di-scFv, tandem tri-scFv, "multi-antibodies" (such as tri- or tetra-antibodies), and single-domain antibodies such as nanobodies or monovariable domain antibodies containing only one variable domain, which may be VHH, VH, or VL that specifically binds to an antigen or epitope independently of other V regions or domains. As used herein, the terms “single-chain Fv,” “single-chain antibody,” or “scFv” refer to single-peptide chain antibody fragments that contain variable regions from both the heavy and light chains but lack constant regions. Typically, single-chain antibodies further include a polypeptide linker between the VH and VL domains, which allows them to form the desired structure that will permit antigen binding. Single-chain antibodies are discussed in detail in: Pluckthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994). Various methods for generating 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, the single-chain antibody may also be bispecific, multispecific, human and / or humanized, and / or synthetic.

[0368] Furthermore, the term "antibody construct" is defined to include monovalent, divalent, and polyvalent / multivalent constructs, and therefore includes bispecific constructs that specifically bind to only two antigen structures, and multispecific constructs that specifically bind to more than two (e.g., three, four, or more) antigen structures through different binding domains. Additionally, the term "antibody construct" is defined to include molecules consisting of only one polypeptide chain and molecules consisting of more than one polypeptide chain, which can be identical (homodimer, homotrimer, or homooligomer) or different (heterodimer, heterotrimer, or heterooligomer). Examples of the antibodies and variants or their derivatives identified above are particularly described in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988), and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Dübel, Antibody Engineering, Springer, 2nd edition, 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press, 2009.

[0369] As used herein, the term "bispecific" refers to an antibody construct that is "at least bispecific," meaning that the antibody construct contains at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (here: MUC17), and the second binding domain binds to another antigen or target (here: CD3). Therefore, the antibody construct according to the invention contains specificity against at least two different antigens or targets. For example, the first domain preferably does not bind to the extracellular epitopes of one or more of the species described herein. The term "target cell surface antigen" refers to an antigenic structure expressed by a cell and present on the cell surface such that it is accessible to the antibody construct as described herein. It can be a protein, preferably the extracellular portion of a protein, or a carbohydrate structure, preferably the carbohydrate structure of a protein, such as a glycoprotein. It is preferably a tumor antigen. The term "bispecific antibody construct" of the invention also encompasses multispecific antibody constructs, such as trispecific antibody constructs, which include three binding domains, or constructs having more than three (e.g., four, five…) specificities.

[0370] Given that the antibody constructs applied according to the present invention are (at least) bispecific, they are not naturally occurring and are distinctly different from naturally occurring products. Therefore, a “bispecific” antibody construct or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sides with different specificities. Bispecific antibody constructs can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. [Clinical Experimental Immunology] 79:315-321 (1990). The at least two binding domains and variable domains (VH / VL) of the antibody construct may or may not contain peptide linkers (spacer peptides). According to the present invention, the term “peptide linker” comprises an amino acid sequence through which 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 are linked to each other. Peptide linkers can also be used to fuse a third domain with other domains of the antibody construct. A fundamental technical feature of such a peptide linker is that it does not contain any polymerization activity. Suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233 or WO 88 / 09344. Peptide linkers can also be used to attach other domains or modules or regions (such as half-life extension domains) to antibody constructs.

[0371] The antibody construct is preferably a "virtually generated antibody construct." This term refers to an antibody construct as defined above, in which all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection process, such as in vitro phage display, protein microarray, or any other method in which the ability of a candidate sequence to bind to an antigen can be tested. Therefore, this term preferably excludes sequences generated solely through genomic rearrangement in the immune cells of an animal. A "recombinant antibody" is an antibody produced using recombinant DNA technology or genetic engineering.

[0372] As used herein, the term "monoclonal antibody" (mAb) or monoclonal antibody construct refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprising that population are identical except for possibly small amounts of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies exhibit high specificity against a single antigenic side or determinant on an antigen, compared to conventional (polyclonal) antibody formulations that typically comprise different antibodies targeting different determinants (or epitopes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized via hybridoma culture and thus free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. To prepare monoclonal antibodies, any technique that provides antibodies produced through continuous cell line culture can be used. For example, monoclonal antibodies to be used can be prepared first by a hybridoma method described by Koehler et al., Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Examples of other techniques for producing human monoclonal antibodies include trigeneous hybridoma technology, human B-cell hybridoma technology (Kozbor, Immunology Today, 4 (1983), 72), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Hybridomas can then be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis such as Biacore™ to identify one or more hybridomas that produce antibodies that specifically bind to a specified antigen. Any form of relevant antigen can be used as an immunogen, such as recombinant antigens, naturally occurring forms, any variants or fragments thereof, and their antigenic peptides. For example, surface plasmon resonance, as employed in the Biacore system, can be used to increase the efficiency of phage antibodies binding to epitopes of target cell surface antigens (Schier, Human Antibodies Hybridomas, 7(1996), 97-105; Malmborg, J. Immunol. Methods, 183(1995), 7-13). Another exemplary method for preparing monoclonal antibodies involves screening protein expression libraries, such as phage display or ribosome display libraries.Phage display is described, for example, in the following: Ladner et al., U.S. Patent 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).

[0373] In addition to using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (e.g., mice, hamsters, rabbits, or rats). In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, a large fragment of a human Ig (immunoglobulin) gene locus can be used to engineer mouse strains that are deficient in antibody production. By using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with desired specificity can be generated and selected. See, for example, XENOMOUSE™; Green et al. (1994) Nature Genetics 7:13-21; US ​​2003-0070185; WO 96 / 34096 and WO96 / 33735.

[0374] Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, such as humanization, deimmunization, and chimerism. Examples of modified antibody constructs include humanized variants of non-human antibodies, “affinity-matured” antibodies (see, for example, Hawkins et al. J. Mol. Biol. [Journal of Molecular Biology] 254, 889-896 (1992) and Lowman et al., Biochemistry [Biochemistry] 30, 10832-10837 (1991)) and antibody mutants with altered functions of one or more effectors (see, for example, U.S. Patent 5,648,260, Kontermann and Dübel (2010, cited above) and Little (2009, cited above).

[0375] In immunology, affinity maturation is a process through which B cells produce antibodies with increased affinity for antigens during an immune response. Through repeated exposure to the same antigen, the host will produce antibodies with successively higher affinities. Similar to the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations are introduced within the CDR using radiation, chemical mutagens, or error-prone PCR. Furthermore, genetic diversity can be increased through strand shuffling. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with low nanomolar affinities.

[0376] Preferred types of amino acid substitution variations in antibody constructs involve substituting one or more hypervariable residues of the parent antibody (e.g., humanized or human antibody). Typically, one or more resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such substituted variants includes phage-displayed affinity maturation. Briefly, several hypervariable side ends (e.g., 6-7 side ends) are mutated to generate all possible amino acid substitutions at each side end. The resulting antibody variants are displayed monovalently from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable side ends for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively or additionally, analyzing the crystal structure of the antigen-antibody complex to identify contact points between the binding domain and, for example, human MUC17 may be advantageous. Such contact residues and adjacent residues are candidates for substitution according to the techniques described herein. Once such variants are generated, the variant group is screened as described herein, and antibodies that exhibit superior properties in one or more relevant assays can be selected for further development.

[0377] The monoclonal antibodies and antibody constructs applied according to the present invention specifically include “chimeric” antibodies (immunoglobulins) wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81:6851-6855 (1984)). Target chimeric antibodies include “primate-derived” antibodies, which contain a variable domain antigen-binding sequence derived from non-human primates (e.g., Old World monkeys, apes, etc.) and a human constant region sequence. Various methods for preparing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. ScLU. SA [Proceedings of the National Academy of Sciences] 81:6851, 1985; Takeda et al., Nature [Nature] 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., EP0171496; EP 0173494; and GB 2177096.

[0378] Antibodies, antibody constructs, antibody fragments, or antibody variants can also be modified by specifically deleting human T-cell epitopes using methods disclosed, for example, in WO 98 / 52976 or WO00 / 34317 (referred to as “deimmunization” methods). In short, the heavy and light chain variable domains of antibodies can be analyzed against peptides that bind to class II MHC; 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, and in addition, databases of human MHC class II binding peptides, as described in WO 98 / 52976 and WO 00 / 34317, can be searched against motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes and thus constitute potential T-cell epitopes. Detected potential T-cell epitopes can be eliminated by substituting a few amino acid residues in the variable domain, or preferably by a single amino acid substitution. Typically, conserved substitutions are performed. Usually, but not exclusively, amino acids common to positions in human immunoglobulin sequences can be used. Human sequences are disclosed, for example, in: Tomlinson et al. (1992) J. MoI. Biol. [Journal of Cell Biology] 227:776-798; Cook, GP et al. (1995) Immunol. Today [Contemporary Immunology] Vol. 16(5):237-242; and Tomlinson et al. (1995) EMBO J. [Journal of the European Society for Molecular Biology] 14:14:4628-4638. The V BASE catalog provides a comprehensive catalog of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as sources of human sequences, for example, for the frame region and CDR. A shared human frame area may also be used, such as that described in U.S. Patent No. 6,300,064.

[0379] "Humanized" antibodies, antibody constructs, variants, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) are antibodies or immunoglobulins with a predominantly human sequence containing one or more minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the hypervariable region (also known as the CDR) of the receptor are replaced by residues from the hypervariable region (donor antibody) of a non-human (e.g., rodent) species (such as mouse, rat, hamster, or rabbit) with the desired specificity, affinity, and ability. 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 contain residues not found in either the receptor antibody or the donor antibody. These modifications are made to further improve and optimize antibody performance. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc) 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). Humanized antibodies or fragments thereof can be generated by replacing sequences of Fv variable domains that are 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 US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. Those methods involve isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of the variable domain of immunoglobulin Fv from at least one of the heavy or light chains. This nucleic acid can be obtained from hybridomas and from other sources as described above by generating antibodies against a predetermined target. The recombinant DNA encoding the humanized antibody molecule can then be cloned into a suitable expression vector. Humanized antibodies can also be generated using transgenic animals such as mice expressing human heavy and light chain genes but not endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR transplantation method (US Patent No. 5,225,539) that can be used to prepare the humanized antibodies described herein. All CDRs of a particular human antibody may be replaced with at least a portion of non-human CDRs, or only some CDRs may be replaced with non-human CDRs.Only the number of CDRs required for the humanized antibody to bind to the predetermined antigen needs to be replaced. Humanized antibodies can be optimized by introducing conserved substitutions, shared sequence substitutions, germline substitutions, and / or reversion mutations. Such modified immunoglobulin molecules can be prepared using 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).

[0380] 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) (cited above). The human antibodies, antibody constructs, or binding domains of the present invention may contain, for example, amino acid residues not encoded by human germline immunoglobulin sequences in CDRs, particularly CDR3 (e.g., mutations introduced by in vitro random or flanking-specific mutagenesis or by in vivo somatic mutations). Human antibodies, antibody constructs, or binding domains may have at least one, two, three, four, five, or more sites where amino acid residues are substituted, which are not encoded by human germline immunoglobulin sequences. However, the definitions of human antibodies, antibody constructs, and binding domains as used herein also consider “fully human antibodies,” which contain only human antibody sequences that are not artificially and / or genetically altered, such as those derived using techniques or systems such as Xenomouse. Preferably, the “fully human antibody” does not include amino acid residues not encoded by human immunoglobulin sequences.

[0381] The antibody constructs applied according to the present invention are “isolated” or “substantially pure” antibody constructs. When used to describe the antibody constructs disclosed herein, “isolated” or “substantially pure” means that the antibody construct has been identified, isolated, and / or recovered from the components of its production environment. Preferably, the antibody construct is free from or substantially unrelated to all other components from its production environment. Contaminating components of its production environment (such as those produced by recombinant transfected cells) are typically substances that interfere with the diagnostic or therapeutic use of the peptide and may include enzymes, hormones, and other proteins or non-protein solutes. The antibody construct may, for example, constitute at least about 5% or at least about 50% by weight of the total protein in a given sample. It should be understood that the isolated protein may, depending on the circumstances, constitute from 5% to 99.9% by weight of the total protein content. Significantly higher concentrations of the peptide can be prepared by using an inducible promoter or a high-expression promoter, resulting in increased concentration levels of the peptide. This definition includes the production of antibody constructs in a variety of organisms and / or host cells known in the art. In a preferred embodiment, the antibody construct (1) is purified to a degree sufficient to obtain at least 15 N-terminal or internal amino acid residues using a spin-cup sequencer, or (2) is purified to homogenization by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. However, typically, the isolated antibody construct is prepared by at least one purification step.

[0382] The term "binding domain" characterizes a domain that binds to / interacts with / recognizes a given target epitope or a given target side end (specifically) on a target molecule (antigen), wherein the target molecule or antigen is 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, such as a full-length or intact immunoglobulin molecule, and / or extracted from the variable heavy chain (VH) and / or variable light chain (VL) domains of the antibody or its fragments. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The second binding domain preferably also includes the minimum structural requirements of the antibody that allow target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). It is envisioned that the first binding domain and / or the second binding domain are generated or available through phage display or library screening methods, rather than by transplanting CDR sequences from a pre-existing (monoclonal) antibody into a scaffold.

[0383] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may include protein portions and non-protein portions (e.g., chemical linkers or chemical cross-linking agents, such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments, and peptides typically having fewer than 30 amino acids) comprise two or more amino acids coupled to each other via covalent peptide bonds (forming an amino acid chain).

[0384] As used herein, the term "peptide" describes a group of molecules that typically consists of more than 30 amino acids. Peptides can further form polymers, such as dimers, trimers, and higher oligomers, i.e., composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc., can be identical or different. Therefore, the corresponding high-order structures of such polymers are called homo- or hetero-dimers, homo- or hetero-trimers, etc. An example of a hetero-polymer is an antibody molecule, which in its naturally occurring 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, where the modification is achieved, for example, through post-translational modifications (such as glycosylation, acetylation, phosphorylation, etc.). When mentioned herein, "peptide," "polypeptide," or "protein" can also be chemically modified, such as polyethylene glycol-modified. Such modifications are well known in the art and are described below.

[0385] Preferably, the binding domains that bind to MUC17 and / or CD3ε are human binding domains. Antibodies and antibody constructs containing at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs having non-human (such as rodents, e.g., mice, rats, hamsters, or rabbits) variable and / or constant regions. The presence of such rodent-derived proteins can lead to rapid clearance of the antibody or antibody construct, or can lead to the generation of an immune response against the antibody or antibody construct in the patient. To avoid using rodent-derived antibodies or antibody constructs, human or fully human antibody / antibody constructs can be generated by introducing human antibody function into rodents to produce fully human antibodies in rodents.

[0386] The ability to clone and reconstruct megabase-sized human loci in yeast artificial chromosomes (YACs) and introduce them into mouse lines provides a powerful method for elucidating the functional components of very large or coarsely located loci and for generating useful human disease models. Furthermore, replacing mouse loci with their human equivalents using this technique can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0387] A key practical application of this strategy is the "humanization" of the mouse humoral immune system. Introducing human immunoglobulin (Ig) loci into mice in which endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms of programmed antibody expression and assembly, as well as their role in B cell development. Furthermore, this strategy can provide an ideal source for the production of fully human monoclonal antibodies (mAbs)—a significant milestone contributing to the prospect of antibody therapy in human diseases. Fully human antibodies or antibody constructs are expected to minimize the immunogenicity and allergic responses inherent in mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of administered antibody / antibody constructs. The use of fully human antibodies or antibody constructs can be expected to offer significant advantages in treating chronic and relapsing human diseases such as inflammation, autoimmune diseases, and cancer that require repeated compound administration. One approach to achieving this is to engineer mouse strains deficient in antibody production with large fragments of human Ig loci, which are expected to produce large amounts of human antibodies in the absence of mouse antibodies. Large human Ig fragments will preserve large variable genetic diversity and appropriate regulation of antibody production and expression. By leveraging the antibody diversification and selection mechanisms of mice, as well as their lack of immune tolerance to human proteins, regenerated human antibody repositories in these mouse strains should produce high-affinity antibodies against any antigen of interest, including human antigens. Antigen-specific human mAbs with desired specificity can be readily generated and selected using hybridoma technology. This general strategy is relevant to first-generation XenoMouse mouse strains (see Green et al., Nature Genetics, 7:13-21 (1994)). XenoMouse strains were engineered with YACs containing germline fragments of 245 kb and 190 kb, respectively, of human heavy chain and κ light chain loci, containing core variable and constant region sequences. YACs containing human Ig were demonstrated to be compatible with mouse systems regarding antibody rearrangements and expression, and to be able to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development, produce adult-like human repositories of fully human antibodies, and generate antigen-specific human mAbs. These results also demonstrate that introducing human Ig loci containing a greater number of V genes, additional regulatory elements, and a larger portion of the human Ig constant region can substantially reproduce a complete repertoire that characterizes the humoral response to infection and immunity. The work of Green et al. has recently been extended to introducing human antibody repertoires greater than approximately 80% by introducing megabase-sized germline YAC fragments of human heavy chain and κ light chain loci, respectively. See Mendez et al., Nature Genetics 15:146-156 (1997) and U.S. Patent Application Serial No. 08 / 759,620.The creation of XenoMouse animals is further discussed and explained in the following: U.S. Patent Application 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 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, serial numbers 08 / 724,752, and 08 / 759,620; and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598; and Japanese Patent Nos. 3068180B2, 3068506B2, and 3068507B2. 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.

[0388] In another approach, other companies (including GenPharm International) have utilized the “microlobe” approach. In the microlobe approach, a foreign Ig locus is mimicked by including a single gene from an 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 γ constant region) form a construct for insertion into an animal. The method is described in the following: U.S. Patent Nos. 5,545,807 (Surani et al.) and 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; 5,874,299; and 6,255,458 (Lonberg and Kay, respectively); U.S. Patent Nos. 5,591,669 and 6,023,010 of Krimpenfort and Berns; and Berns et al. U.S. Patent Nos. 5,612,205, 5,721,367, and 5,789,215, and U.S. Patent Nos. 5,643,763 of Choi and Dunn, and U.S. Patent Application Serial 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 of GenPharm International. See EP0546073 B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, and U.S. Patent No. 5,981,175. Also see Taylor et al. (1992) and (1994), Chen et al. (1993), Tuaillon et al. (1993) and (1995), Choi et al. (1993), Lonberg et al. (1994), and Fishwild et al. (1996).

[0389] Kirin has also demonstrated the generation of human antibodies from mice in which large chromosomal fragments or entire chromosomes have been introduced via microcell fusion. See European patent applications 773 288 and 843 961. Xenerex Biosciences is developing a technology that may generate human antibodies. In this technology, SCID mice are reconstructed with human lymphocytes (e.g., B and / or T cells). The mice are then immunized with an antigen, and an immune response against the antigen can be generated. See U.S. Patents 5,476,996; 5,698,767; and 5,958,765.

[0390] Human anti-mouse antibody (HAMA) responses have led the industry to develop chimeric antibodies or other humanized antibodies. However, certain human anti-chimeric antibody (HACA) responses are expected, particularly with long-term or multiple-dose use of antibodies. Therefore, it is desirable to provide antibody constructs containing a human binding domain against MUC17 and a human binding domain against CD3ε to eliminate the problems and / or effects of HAMA or HACA responses.

[0391] The terms “(selective) binding”, “(specific) binding”, “(specific) recognition”, “(specific) targeting” and “(specific) reaction” mean, according to the present invention, that the binding domain interacts or specifically interacts with a given epitope or a given target side end on a target molecule (antigen) (here: MUC17 and CD3ε, respectively).

[0392] The term "epitope" refers to a side of 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 is sometimes referred to herein as an "antigen structure" or "antigen determinant." Thus, the binding domain is the "antigen interaction side." The binding / interaction is also understood to define "specific recognition." An epitope can be formed by consecutive amino acids or by discontinuous amino acids juxtaposed through the tertiary folding of a protein. A "linear epitope" is an epitope in which the primary sequence of amino acids contains the recognized epitope. Linear epitopes typically comprise at least 3 or at least 4, and more usually at least 5 or at least 6 or at least 7, for example, about 8 to about 10 amino acids in a unique sequence. In contrast to linear epitopes, a "conformal epitope" is an epitope in which the primary sequence of amino acids constituting the epitope is not the only defining component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the binding domain). Typically, conformational epitopes contain an increased number of amino acids compared to linear epitopes. Regarding the recognition of conformational epitopes, binding domains recognize the three-dimensional structure of an antigen, preferably a peptide or protein, or a fragment thereof (in the context of this invention, an antigen structure containing a binding domain is included within an antigen protein on the surface 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, enabling the antibody to recognize the epitope. Methods for determining epitope conformation include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy. The following describes a method for epitope mapping: when a region (continuous amino acid extension) in a human MUC17 protein is exchanged or replaced with a corresponding region in a non-human, non-primate MUC17 (e.g., mouse MUC17, but others such as chicken, rat, hamster, rabbit, etc. are also possible), a decrease in binding of the binding domain is expected, unless the binding domain is cross-reactive with the non-human, non-primate MUC17 used. The reduction in binding to the corresponding region in the human MUC17 protein 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%, thereby setting the binding to the corresponding region in the human MUC17 protein to 100%. It is envisioned that the aforementioned human MUC17 / non-human MUC17 chimera is expressed in CHO cells. It is also envisioned that the human MUC17 / non-human MUC17 chimera fuses with the transmembrane domains and / or cytoplasmic domains of different membrane-bound proteins (such as EpCAM).

[0393] In alternative or additional methods for epitope mapping, several truncated forms of human MUC17 extracellular domains can be generated to identify specific regions recognized by the binding domain. In these truncated forms, different extracellular MUC17 domains / subdomains or regions are progressively deleted starting from the N-terminus. It is envisioned that the truncated MUC17 forms can be expressed in CHO cells. It is also envisioned that the truncated MUC17 forms can fuse with transmembrane domains and / or cytoplasmic domains of different membrane-binding proteins, such as EpCAM. It is also envisioned that the truncated MUC17 forms may encompass 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 envisioned that the truncated MUC17 forms may encompass a v5 domain at their N-terminus (after the signal peptide), which would allow verification of their correct expression on the cell surface. Reduced or lost binding is expected for those truncated MUC17 forms that no longer encompass the MUC17 region recognized by the binding domain. The binding 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%, thereby setting the binding to the whole human MUC17 protein (or its extracellular region or domain) to 100%.

[0394] Another method for determining the contribution of specific residues of MUC17 to the recognition of antibody constructs or binding domains is alanine scanning (see, for example, Morrison KL and Weiss GA. Cur Opin Chem Biol. [New Insights in Chemical Biology] 2001, June; 5(3):302-7), where each residue to be analyzed is replaced with alanine, for example, via site-directed mutagenesis. Alanine is used because it has a non-giant, chemically inert methyl functional group, but still mimics the secondary structure references of many other amino acids. In cases where the size of conserved mutant residues is required, giant amino acids (such as valine or leucine) can sometimes be used. Alanine scanning is a well-established technique that has been used for a long time.

[0395] The interaction between the binding domain and the epitope or epitope-containing region means that the binding domain exhibits considerable affinity for the epitope and / or epitope-containing region on a specific protein or antigen (here: MUC17 and CD3, respectively), and generally does not exhibit significant reactivity with proteins or antigens other than MUC17 or CD3 unless otherwise stated. "Considerable affinity" includes affinity at approximately 10... -6 M(KD) or stronger affinity binding. Preferably, when the binding affinity is about 10... -12 Up to 10 -8 M, 10 -12 Up to 10 -9 M, 10 -12 Up to 10-10 M, 10 -11 Up to 10 -8 M, preferably about 10 -11 Up to 10 -9 When M is present, binding is considered specific. Whether a binding domain specifically reacts with or binds to a target can be readily tested, particularly by comparing the reaction of the binding domain with a target protein or antigen and the reaction of the binding domain with a protein or antigen other than MUC17 or CD3. Preferably, the binding domains of the present invention substantially 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). It is envisioned that the antibody constructs according to the present invention are characterized by superior affinity characteristics compared to other HLE forms. Therefore, this superior affinity indicates a prolonged half-life in vivo. The longer half-life of the antibody constructs according to the present invention can reduce the duration and frequency of administration that typically contributes to improving patient compliance. This is particularly important because the antibody constructs of the present invention are especially beneficial for highly debilitated or even multiple cancer patients.

[0396] The terms “substantially / truly non-binding” or “cannot bind” mean that the binding domain of the present invention does not bind to proteins or antigens other than MUC17 or CD3, that is, it does not show reactivity of more than 30%, preferably no more than 20%, more preferably no more than 10%, particularly preferably no more than 9%, 8%, 7%, 6% or 5% with proteins or antigens other than MUC17 or CD3, thereby setting the binding with MUC17 or CD3 as 100% respectively.

[0397] Specific binding is believed to be influenced by specific motifs in the binding domain and the amino acid sequence of the antigen. Therefore, binding is achieved as a result of their primary, secondary, and / or tertiary structures, as well as secondary modifications of these structures. The specific interaction between the antigen-interacting side and its specific antigen can lead to simple binding of the side to the antigen. Furthermore, the specific interaction between the antigen-interacting side and its specific antigen can alternatively or additionally lead to signal initiation, for example, due to induced changes in antigen conformation, antigen oligomerization, etc.

[0398] The term "variable" refers to the portion of an antibody or immunoglobulin domain that exhibits sequence variability and participates in determining the specificity and binding affinity of a particular antibody (i.e., "one or more variable domains"). The pairing of variable heavy chains (VH) and variable light chains (VL) together forms a single antigen-binding site. The variability is not uniformly distributed throughout the variable domains of an antibody; it 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) and provide a scaffold for the six CDRs in three-dimensional space to form the antigen-binding surface. The naturally occurring variable domains of the heavy and light chains each contain four FRM regions (FR1, FR2, FR3, and FR4), which primarily utilize the β-sheet conformation and are connected by three hypervariable regions that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are closely clustered together by the FRM and together with the hypervariable regions from the other chain, they contribute to the formation of antigen-binding side ends (see Kabat et al., cited above).

[0399] The term “CDR” and its plural “CDRs” refer to the complementary determinant regions of three binding features constituting the light chain variable regions (CDR-L1, CDR-L2, and CDR-L3) and three binding features constituting the heavy chain variable regions (CDR-H1, CDR-H2, and CDR-H3). CDRs contain most of the residues responsible for the antibody-antigen specific interaction and thus contribute to the functional activity of the antibody molecule: they are the major determinants of antigen specificity. The precisely defined boundaries and lengths of CDRs are subject to different classification and numbering systems. Therefore, CDRs can be referenced by Kabat, Chothia, contact, or any other boundary definition (including the numbering system described herein). Despite the different boundaries, each of these systems has a degree of overlap in constituting the so-called “hypervariate regions” within the variable sequence. Therefore, CDR definitions according to these systems can differ in length and boundary regions relative to adjacent frame regions. See, for example, Kabat (a method based on cross-species sequence variability), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., cited above; Chothia et al., J. MoI. Biol, 1987, 196: 901-917; and MacCallum et al., J. MoI. Biol, 1996, 262: 732). Another standard for characterizing antigen-binding flanking ends is the AbM definition used by the AbM antibody modeling software from Oxford Molecular. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains in: Antibody Engineering Lab Manual (edited below: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Regarding the use of two residue identification techniques to define overlapping regions rather than identical regions, they can be combined to define heterozygous CDRs. However, numbering according to the so-called Kabat system is preferred. Typically, CDRs form ring structures that can be classified as canonical structures. The term "canonical structure" refers to the main chain conformation adopted by the antigen-binding (CDR) ring. From comparative structural studies, five of the six antigen-binding rings have been found to have only a limited library of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone.Therefore, the corresponding loops between antibodies can have very similar three-dimensional structures, despite high amino acid sequence variability in most parts of the loop (Chothia and Lesk, J. MoI. Biol. [Journal of Molecular Biology], 1987, 196:901; Chothia et al., Nature [Nature], 1989, 342:877; Martin and Thornton, J. MoI. Biol. [Journal of Molecular Biology], 1996, 263:800). Furthermore, there is a relationship between the adopted loop structure and its surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop and within the conserved framework (i.e., outside the loop). Therefore, the assignment of a particular canonical class can be based on the presence of these key amino acid residues.

[0400] The term "canonical structure" can also include considerations regarding the linear sequence of the antibody, such as those cataloged by Kabat (Kabat et al., cited above). The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues of variable domains of antibodies in a consistent manner and is the preferred scheme for the application of this invention, as also mentioned elsewhere herein. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al. and / or revealed by other techniques, such as crystallography and two- or three-dimensional computational modeling. Thus, a given antibody sequence can be placed in a canonical category, which in particular allows for the identification of appropriate chassis sequences (e.g., based on the expectation of including multiple canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations, as described by Chothia et al. (cited above), and their implications for interpreting canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known in the art. For a review of antibody structures, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, edited by Harlow et al., 1988.

[0401] The CDR3 of the light chain, and especially the CDR3 of the heavy chain, can constitute the most important determinants of antigen binding within the variable regions of both the light and heavy chains. In some antibody constructs, the heavy chain CDR3 appears to constitute the primary contact region between the antigen and antibody. In vitro selection schemes that modify CDR3 individually can be used to alter antibody binding properties or determine which residues contribute to antigen binding. Therefore, CDR3 is typically the largest source of molecular diversity within the antibody binding side. For example, H3 can be as short as two amino acid residues or more than 26 amino acids.

[0402] In classic full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by a covalent disulfide bond, while 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 named CH1. The constant (“C”) domain does not directly participate in antigen binding but exhibits various effector functions, such as antibody-dependent, cell-mediated cytotoxicity, and complement activation. The Fc region of an antibody is included within the heavy chain constant domain and can, for example, interact with Fc receptors located on the cell surface.

[0403] The sequences of antibody genes are highly altered after assembly and somatic mutation, and it is estimated that these altered genes encode 10. 10 Different antibody molecules (Immunoglobulin Genes, 2nd Edition, edited by Jonio et al., Academic Press, San Diego, CA, 1995). Therefore, the immune system provides an immunoglobulin repertoire. The term "repertoire" refers to at least one nucleotide sequence, wholly or partially derived from at least one sequence encoding at least one immunoglobulin. One or more sequences can be generated by in vivo rearrangement of the V, D, and J segments of the heavy chain and the V and J segments of the light chain. Alternatively, one or more sequences can be generated from cells in response to rearrangement (e.g., in vitro stimulation). Alternatively, some or all of one or more sequences can be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, for example, U.S. Patent 5,565,332. A repertoire may include only one sequence or may include multiple sequences, including sequences from a genetic diversity set.

[0404] The term "Fc moiety" or "Fc monomer" in this invention refers to a polypeptide comprising at least one domain functional with a CH2 domain and at least one domain functional with a CH3 domain of an immunoglobulin molecule. It is evident from the term "Fc monomer" that a polypeptide comprising those CH domains is a "polypeptide monomer." An Fc monomer can be a polypeptide comprising at least a fragment of an immunoglobulin constant region excluding the first constant region immunoglobulin domain (CH1) of the heavy chain, but retaining at least a functional portion of a CH2 domain and a functional portion of a CH3 domain, wherein the CH2 domain is at the amino terminus of the CH3 domain. In a preferred aspect of this definition, the Fc monomer can be a polypeptide constant region comprising a portion of an Ig-Fc hinge region, a CH2 region, and a CH3 region, wherein the hinge region is at the amino terminus of the CH2 domain. It is envisioned that the hinge region of the invention promotes dimerization. For example, but not limited to, such Fc polypeptide molecules can be obtained by digesting the immunoglobulin region with papain (generating, of course, a dimer of two Fc polypeptides). In another aspect of this definition, the Fc monomer can be a polypeptide region comprising portions of both the CH2 and CH3 regions. For example, but not limited to, such Fc polypeptide molecules can be obtained by digesting immunoglobulin molecules with pepsin. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the following Fc polypeptide sequences: IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region. (See, for example, Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because there are some variations among immunoglobulins, and only for clarity, the Fc monomer refers to the last two heavy chain constant regions of the immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant regions of the immunoglobulin domains of IgE and IgM.

[0405] As mentioned above, Fc monomers may also include flexible hinges at the N-termini of these domains. For IgA and IgM, Fc monomers may include J chains. For IgG, the Fc moiety comprises immunoglobulin domains CH2 and CH3, and a hinge between the first two domains and CH2. Although the boundaries of the Fc moiety can vary, an example of a human IgG heavy chain Fc moiety comprising a functional hinge, CH2, and CH3 domains can be defined, for example, as P476 comprising residue D231 (a residue of the hinge domain—corresponding to D234 in Table 2 below) to the carboxyl terminus of the CH3 domain, respectively, L476 (for IgG4), where the numbering is based on Kabat. Two Fc moieties or Fc monomers fused together via peptide linkers define a third domain of the antibody construct of the present invention, which may also be defined as an scFc domain. Contemplate that Fc monomers fused together separately as disclosed herein are contained only in the third domain of the antibody construct.

[0406] Table 2: Kabat numbers of amino acid residues in the hinge region

[0407]

[0408]

[0409] The location and sequence of the IgG CH2 and IgG CD3 domains can be identified by analogy using the Kabat numbers listed in Table 3:

[0410] Table 3: Kabat numbers of amino acid residues in the CH2 and CH3 regions of IgG

[0411]

[0412] In one embodiment of the invention, the amino acid residues highlighted in bold are removed from the CH3 domain of the first or both Fc monomers.

[0413] The peptide linker where the peptide monomers of the third domain (“Fc moiety” or “Fc monomer”) are fused together preferably contains at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker contains at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). Even more preferably, the linker contains up to 40 amino acid residues, more preferably up to 35 amino acid residues, and most preferably exactly 30 amino acid residues.

[0414] When using a linker to fuse a first domain with a second domain or to fuse a first or second domain with a third domain, the linker preferably has a length and sequence sufficient to ensure that each of the first and second domains can independently retain its differential binding specificity. For peptide linkers connecting at least two binding domains (or two variable domains) in an antibody construct, those containing only a small number of amino acid residues (e.g., 12 amino acid residues or less) are preferred. Therefore, peptide linkers with 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. Preferred examples of peptide linkers for fusing the first and second domains are depicted in SEQ ID NO:10. A preferred linker example for fusing the second and third domains is the (Gly)4-linker, also known as the G4-linker. Features of peptide linkers that do not promote secondary structures are known in the art and described, for example, by 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 connection between the domains can be provided, for example, by genetic engineering, as described in the examples. Methods for preparing fused and operably linked bispecific single-stranded 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).

[0415] According to a particularly preferred embodiment, the first and second domains of the antibody construct of the present invention are “bispecific single-chain antibody constructs,” more preferably bispecific “single-chain Fv” (scFv). Although the two domains, VL and VH, of the Fv fragment are encoded by independent genes, these two domains can be joined by a synthetic linker using a recombinant approach, as described above, which allows them to be prepared as a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as that of complete or full-length antibodies. Thus, a single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, typically using a short linker peptide of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is typically enriched with glycine for flexibility and with serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin.

[0416] Bispecific single-chain antibody constructs are known in the art and described in the following: WO 99 / 54440; Mack, J. Immunol. [Journal of Immunology] (1997), 158, 3965-3970; Mack, PNAS [Proceedings of the National Academy of Sciences of the United States of America], (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother. [Cancer Immunology and Immunotherapy], (1997), 45, 193-197; Blood, (2000), 95, 6, 2098-2103; Brühl, Immunol, (2001), 166, 2420-2426; Kipriyanov, J. Mol. Biol, (1999), 293, 41-56. Single-chain antibody constructs that specifically recognize one or more selected targets can be generated using the techniques described for generating single-chain antibodies (see in particular U.S. Patent 4,946,778; Kontermann and Dübel (2010), cited above; and Little (2009), cited above).

[0417] Bivalent (also known as divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv having the form (scFv)2) can be engineered by linking two scFv molecules (e.g., using a linker as described above). If the two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule will preferably be called bivalent (i.e., having two valences for the same target epitope). If the two scFv molecules have different binding specificities, the resulting (scFv)2 molecule will preferably be called bispecific. Linking can be performed by generating a tandem scFv by producing a single peptide chain with two VH regions and two VL regions (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to generate scFv molecules with linker peptides that are too short for the two variable regions to fold together (e.g., about five amino acids), thus forcing the scFv to dimerize. This type is called a biantibody (see, for example, Hollinger, Philipp et al., (July 1993) PNAS [Proceedings of the National Academy of Sciences] 90(14):6444-8).

[0418] The first, second, or first and second domains may comprise a single-domain antibody, which is a variable domain or at least a CDR of the single-domain antibody. A single-domain antibody contains only one (monomer) antibody variable domain that can selectively bind to a specific antigen independently of the other V regions or domains. First single-domain antibodies are engineered from heavy-chain antibodies found in camels, and these are referred to as V... H H fragment. Cartilaginous fish also possess heavy chain antibodies (IgNARs), from which a substance called V can be obtained. NAR Single-domain antibodies. An alternative approach is to split the dimeric variable domain from common immunoglobulins, such as those from humans or rodents, into monomers, thus obtaining VH or VL as single-domain Abs. Although most research on single-domain antibodies is currently based on heavy-chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single-domain antibodies are so-called sdAbs, nanobodies, or single variable-domain antibodies. Therefore, (single-domain mAb)2 is a monoclonal antibody construct consisting of (at least) two single-domain monoclonal antibodies, each individually selected from the group consisting of: V H V L V H H and V NARThe linker is preferably in the form of a peptide linker. Similarly, the “scFv-single-domain mAb” is a monoclonal antibody construct consisting of at least one single-domain antibody as described above and an scFv molecule as described above. Again, the linker is preferably in the form of a peptide linker.

[0419] Whether an antibody construct competitively binds to another given antibody construct can be measured in competitive assays such as competitive ELISAs or cell-based competitive assays. Avidin-conjugated microparticles (beads) can also be used. Similar to avidin-coated ELISA plates, each of these beads can be used as a substrate on which an assay can be performed when reacted with a biotinylated protein. The antigen is coated onto the beads, and then pre-coated with a first antibody. A second antibody is added, and any additional binding is determined. Possible means for readout include flow cytometry.

[0420] Cell cytotoxicity mediated by antibody constructs can be measured in a variety of 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 to its first domain, the effector cells should also be of macaque origin, such as macaque T cell lines, e.g., 4119LnPx. Target cells should express MUC17 (e.g., human or macaque MUC17) (at least its extracellular domain). Target cells can be cell lines (e.g., stably or transiently transfected with MUC17 (e.g., human or macaque MUC17)). For target cell lines expressing higher levels of MUC17 on their cell surface, EC is expected to be higher. 50 The values ​​are usually lower. The effector cell to target cell (E:T) ratio is typically around 10:1, but this can vary. The cytotoxic activity of the MUC17 bispecific antibody construct can be measured in... 51 Cr release assays (incubation time approximately 18 hours) or FACS-based cytotoxicity assays (incubation time approximately 48 hours) may be performed. Modifications to the assay incubation time (cytotoxic response) are also possible. Other methods for measuring cytotoxicity are well known to those skilled in the art and include MTT or MTS assays, ATP-based assays (including bioluminescent assays), sulforhodamine B (SRB) assays, WST assays, clonogenic assays, and ECIS techniques.

[0421] The cytotoxic activity mediated by the MUC17xCD3 antibody construct is preferably measured in a cell-based cytotoxicity assay. It can also be measured in... 51 Cr- release was measured in the assay. It was determined by EC. 50The value indicates that it corresponds to the half-maximal effective concentration (MCC) of the antibody construct (the concentration of the antibody construct that induces a cytotoxic response midway between baseline and maximum). Preferably, the ECC of the MUC17xCD3 antibody construct is... 50 The value is ≤5000pM or ≤4000pM, more preferably ≤3000pM or ≤2000pM, even more preferably ≤1000pM or ≤500pM, even more preferably ≤400pM or ≤300pM, even more preferably ≤200pM, even more preferably ≤100pM, even more preferably ≤50pM, even more preferably ≤20pM or ≤10pM, and most preferably ≤5pM. Preferably, the MUC17xCD3 antibody construct does not induce / mediate lysis or substantially does not induce / mediate lysis of MUC17 negative cells such as CHO cells. The terms "non-inducing lysis," "substantially non-inducing lysis," "non-mediating lysis," or "substantially non-mediating lysis" mean that the antibody construct of the present invention does not induce or mediate the lysis of more than 30%, preferably no more than 20%, more preferably no more than 10%, particularly preferably no more than 9%, 8%, 7%, 6%, or 5% of MUC17-negative cells, thereby setting the lysis of MUC17-positive human cell lines as 100%. This is generally applicable to antibody constructs at concentrations up to 500 nM. Those skilled in the art know how to measure cell lysis effortlessly.

[0422] The first binding domain and / or the second (or any other) binding domain of the antibody construct of the present invention preferably have cross-species specificity for members of the primate mammal order. Cross-species specific CD3 binding domains are described, for example, in WO 2008 / 119567. According to one embodiment, in addition to binding to human MUC17 and human CD3 respectively, the first binding domain and / or the second binding domain will also bind to primate MUC17 / CD3, including (but not limited to) New World primates (such as marmosets (Callithrix jacchus), woolly tamarins (Saguinus Oedipus), or squirrel monkeys (Saimiri sciureus)), Old World primates (such as baboons and macaques), gibbons, and non-human hominids.

[0423] As used herein, the term "pharmaceutical composition" refers to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention comprise a preferred therapeutically effective amount of one or more antibody constructs of the present invention. 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. The acceptable components of the composition are preferably non-toxic to the recipient at the dose and concentration used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions. These compositions may contain pharmaceutically acceptable carriers. Generally, as used herein, "pharmaceuticalally acceptable carrier" means any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate-buffered saline (PBS) solutions), water, suspensions, emulsions (such as oil / water emulsions), various types of wetting agents, liposomes, dispersion media, and coatings compatible with drug administration, particularly 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. In some embodiments, the pharmaceutical composition may contain formulation materials for purposes of altering, maintaining, or preserving the following aspects of the composition: for example, pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, edited by ARGenrmo, 1990, Mack Publishing Company). In such embodiments, suitable formulation materials may include, but are not limited to:

[0424] • Amino acids, such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine, including charged amino acids, preferably lysine, acetylsine, arginine, glutamate and / or histidine.

[0425] • Antimicrobial agents, such as antibacterial agents and antifungal agents

[0426] • Antioxidants, such as ascorbic acid, methionine, sodium sulfite, or sodium bisulfite;

[0427] • Buffer solutions, buffer systems, and buffering agents for maintaining the composition at physiological pH or slightly lower, preferably at a lower pH of 4.0 to 6.5; examples of buffer solutions are borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids, succinate, phosphate, and histidine; for example, Tris buffer solution with a pH of about 7.0-8.5;

[0428] • Non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate;

[0429] • Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffer media;

[0430] • Biodegradable polymers, such as polyester;

[0431] • Accumulators, such as mannitol or glycine;

[0432] • Chelating agents, such as ethylenediaminetetraacetic acid (EDTA);

[0433] • Isotonic agents and absorption delay agents;

[0434] • Complexing agents, such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin;

[0435] • Filler;

[0436] • Monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol, or xylitol;

[0437] • (Low molecular weight) proteins, peptides, or protein carriers, such as human or bovine serum albumin, gelatin, or immunoglobulins, preferably of human origin;

[0438] • Coloring agents and flavoring agents;

[0439] • Sulfur-containing reducing agents, such as glutathione, lipoic acid, sodium thioacetate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate.

[0440] • Diluent;

[0441] • Emulsifier;

[0442] • Hydrophilic polymers, such as polyvinylpyrrolidone

[0443] • Salt-forming equilibrium ions, such as sodium;

[0444] • Preservatives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc.; examples are: benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide;

[0445] • Metal complexes, such as Zn-protein complexes;

[0446] • Solvents and co-solvents (such as glycerol, propylene glycol, or polyethylene glycol);

[0447] Sugars and sugar alcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbitol, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclic polyols (e.g., inositol), polyethylene glycol; and polyols;

[0448] • Suspension agent;

[0449] Surfactants or wetting agents, such as pluronics, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate), tritium nuclei, tromethamine, lecithin, cholesterol, tyloxacin; the surfactant can be a detergent, preferably with a molecular weight >1.2KD, and / or a polyether, preferably with a molecular weight >3KD; preferred non-limiting examples of detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; preferred non-limiting examples of polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG5000;

[0450] • Stability enhancers, such as sucrose or sorbitol;

[0451] • Tension enhancers, such as alkali metal halides, preferably sodium chloride or potassium chloride; mannitol and sorbitol;

[0452] • Parenteral delivery media, including sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution or non-volatile oils;

[0453] • Intravenous delivery mediators, including fluid and nutritional supplements, and electrolyte supplements (such as those based on Ringer's dextran).

[0454] In addition to the antibody constructs defined herein, the composition may contain other bioactive agents, depending on the intended use of the composition. Such agents may be drugs acting on the gastrointestinal system, drugs acting as cell inhibitors, drugs preventing hyperuricemia, drugs suppressing immune responses (e.g., corticosteroids), drugs modulating inflammatory responses, drugs acting on the circulatory system, and / or agents known in the art such as cytokines. It is also envisioned that the antibody constructs of the present invention be used in co-therapies, i.e., in combination with another anticancer drug. In some embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery form, and required dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, ibid. In some embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibody constructs of the present invention. In some embodiments, the primary medium or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, suitable media or carriers may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, which may be supplemented with other substances commonly found in parenteral administration compositions. Neutral buffered saline or saline mixed with serum albumin are other exemplary media. In some embodiments, the antibody construct compositions of the present invention can be prepared for storage by mixing selected components having the desired purity with optional formulations (REMINGTON'S PHARMACEUTICAL SCIENCES, ibid.) in the form of lyophilized cakes or aqueous solutions. Furthermore, in some embodiments, the antibody constructs of the present invention can be formulated into lyophilized products using suitable excipients (such as sucrose).

[0455] When considering parenteral administration, the therapeutic compositions of the present invention can be provided as a pyrogen-free, parenterally acceptable aqueous solution containing the desired antibody construct of the present invention in a pharmaceutically acceptable medium. A particularly suitable medium for parenteral injection is sterile distilled water, in which the antibody construct of the present invention is formulated into a suitably preserved sterile isotonic solution. In some embodiments, this preparation may include formulating the desired molecules with reagents such as injectable microspheres, bio-erosive particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide controlled or sustained release of the product that can be delivered via accumulated injection. In some embodiments, hyaluronic acid may also be used, which has the effect of promoting duration of circulation. In some embodiments, an implantable drug delivery device may be used to introduce the desired antibody construct.

[0456] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving the formulation of the antibody constructs of the present invention into sustained or controlled delivery / release formulations. Techniques for formulating various other sustained or controlled delivery methods, such as liposome carriers, bioerectable microparticles or porous beads, and accumulation injection, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivering pharmaceutical compositions. Sustained-release formulations may comprise a semi-permeable polymeric matrix in the form of a shaped article, such as a film or microcapsule. Sustained-release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. EP 058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (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, ibid.), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP133,988). Sustained-release compositions may also include liposomes prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 82: 3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949.

[0457] Antibody constructs can also be embedded in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by cohesive techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in coarse-drop emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Oslo, A. (1980).

[0458] Pharmaceutical compositions intended for internal administration are typically provided as sterile formulations. Sterilization can be accomplished by filtration through a sterile filter membrane. When the composition is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions intended for parenteral administration can be stored in lyophilized form or as a solution. Parenteral compositions are typically placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0459] On the other hand, the present invention includes self-buffered antibody construct formulations that can be used as pharmaceutical compositions, as described in International Patent Application WO 06138181 A2 (PCT / US2006 / 022599). There are numerous discussions on protein stabilization and formulation materials, as well as methods useful in this regard, such as Arakawa et al., “Solvent interactions in pharmaceutical formulations,” Pharm Res. 8(3):285-91 (1991); Kendrick et al., “Physical stabilization of proteins inaqueous solution,” RATIONAL DESIGN OF STABLEPROTEIN FORMULATIONS: THEEORY AND PRACTICE, Carpenter and Manning (eds.), Pharmaceutical Biotechnology 13:61-84 (2002); and Randolph et al., “Surfactant-protein interactions,” Pharm Biotechnol. [Pharmaceutical Biotechnology] 13:159-75 (2002), see especially the section relating to the same excipients and processes used in self-buffered protein formulations, particularly concerning protein pharmaceutical products and processes for veterinary and / or human medical use.

[0460] According to some embodiments, salts can be used, for example, to adjust the ionic strength and / or isotonicity of formulations and / or to improve the solubility and / or physical stability of proteins or other components of a composition. It is well known that ions can stabilize the native state of proteins by binding to charged residues on the protein surface and by shielding charged and polar groups in the protein and reducing the strength of their electrostatic interactions, attractive and repulsive interactions. Ions can also stabilize the denatured state of proteins by binding to denatured peptide bonds (--CONHs), particularly in proteins. Furthermore, the interaction of ions with charged and polar groups in proteins can reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubility. The effects of different ion types on proteins vary considerably. Many classification rankings of ions and their effects on proteins that can be used to formulate pharmaceutical compositions have been developed. One example is the Hofmeister series, which ranks ions and polar nonionic solutes by their effects on the conformational stability of proteins in solution. Stable solutes are called "hydrophilic." Destabilizing solutes are called "agnostic." High concentrations of liquefying agents (e.g., >1 molar ammonium sulfate) are typically used to precipitate proteins from solution (“salting out”). Ionizing agents are typically used to denature and / or dissolve proteins (“salting in”). The relative effectiveness of ion pairs in “salting in” and “salting out” defines their place in the Hofmeister series.

[0461] According to various embodiments, free amino acids can be used in antibody construct formulations as build-up agents, stabilizers, antioxidants, and for other standard applications. Lysine, proline, serine, and alanine can be used to stabilize proteins in formulations. Glycine can be used for lyophilization to ensure proper cake structure and properties. In both liquid and lyophilized formulations, arginine can be used to inhibit protein aggregation. Methionine can be used as an antioxidant.

[0462] Polyols include sugars (such as mannitol, sucrose, and sorbitol) and polyhydroxy alcohols (such as glycerol and propylene glycol), as well as polyethylene glycol (PEG) and related substances for the purposes of this discussion. Polyols are lyophilic. They are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols can also be used to modulate the stress of formulations. A polyol that can be used in selected embodiments of the invention is mannitol, which is generally used to ensure the structural stability of the cake in lyophilized formulations. It ensures the structural stability of the cake. Mannitol is often used in conjunction with lyophilization protectants, such as sucrose. Sorbitol and sucrose are preferred agents for modulating stress and are used as stabilizers to prevent freeze-thaw stress during transport or during the preparation of powders in the manufacturing process. Reducing sugars (which contain free aldehyde or ketone groups) such as glucose and lactose can glycosylate surface lysine and arginine residues. Therefore, they are generally not preferred polyols. Furthermore, sugars that form this type of reactive substance, such as sucrose, hydrolyze into fructose and glucose under acidic conditions, thus resulting in saccharification. Such sugars are also not preferred polyols in this respect. PEG can be used to stabilize proteins and as a cryoprotectant, and can be used in this regard.

[0463] Examples of antibody construct formulations further include surfactants. Protein molecules may readily adsorb onto surfaces and readily denature and subsequently aggregate at gas-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These detrimental interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical agitation (such as physical agitation generated during the transport and handling of products). Surfactants are commonly used to prevent, minimize, or reduce surface adsorption. In this respect, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of dehydrated sorbitol polyethoxylates, and poloxamer 188. Surfactants are also commonly used to control protein conformational stability. In this respect, the use of surfactants is protein-specific, as any given surfactant will typically stabilize some proteins while destabilizing others.

[0464] Polysorbates are susceptible to oxidative degradation and often contain sufficient amounts of peroxides to cause oxidation of protein residue side chains, particularly methionine. Therefore, polysorbates should be used with caution and at the lowest effective concentration. In this respect, polysorbates exemplify the general rule that excipients should be used at their lowest effective concentration.

[0465] Examples of antibody construct formulations further include one or more antioxidants. To a certain extent, harmful oxidation of proteins in pharmaceutical formulations can be prevented by maintaining appropriate levels of ambient oxygen and temperature and avoiding light exposure. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. In this regard, useful antioxidants are reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants used in therapeutic protein formulations according to the invention are preferably water-soluble and maintain their activity throughout the product's shelf life. In this regard, EDTA is a preferred antioxidant according to the invention. Antioxidants can damage proteins. For example, reducing agents such as glutathione can in particular disrupt intramolecular disulfide bonds. Therefore, antioxidants are selected, among other things, to eliminate or sufficiently reduce the possibility that they themselves damage proteins in the formulation.

[0466] Formulations may contain metal ions that are protein cofactors and essential for the formation of protein coordination complexes, such as zinc, which is necessary for the formation of certain insulin suspensions. Metal ions can also inhibit some protein-degrading processes. However, metal ions also catalyze 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. +2 Ions (up to 100 mM) can increase the stability of human deoxyribonuclease. However, Mg +2 Mn +2 and Zn +2 This can destabilize rhDNase. Similarly, Ca... +2 and Sr +2 It can stabilize factor VIII, and it can be Mg +2 Mn +2 and Zn +2 Cu +2 and Fe +2 Destabilization, and the aggregation of this stabilizing factor can be achieved through Al. +3 Ion reinforcement.

[0467] Examples of formulations further include one or more preservatives. Preservatives are essential when developing multi-dose parenteral formulations that involve multiple dispensings from the same container. Their primary function is to inhibit microbial growth and ensure sterility throughout the shelf life or intended use of the pharmaceutical product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While the use of preservatives with small-molecule parenteral drugs has a long history, the development of protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect (aggregation) on proteins, and this has become a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single use only. However, when multi-dose formulations become possible, they offer the added advantages of patient convenience and increased marketability. A good example is the preservative for human growth hormone (hGH), where the development of preservative formulations has led to the commercialization of more convenient, multi-purpose injection pens. At least four such pen devices containing preservative hGH formulations are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized, two-chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol. Several aspects need to be considered during the formulation and development of preservation dosage forms. The concentration of effective preservatives in the pharmaceutical product must be optimized. This requires testing a given range of preservative concentrations in the dosage form that imparts antimicrobial efficacy without compromising protein stability.

[0468] As can be expected, developing liquid formulations containing preservatives is more challenging than developing lyophilized formulations. Lyophilized products can be freeze-dried without preservatives and reconstituted with a preservative-containing diluent before use. This shortens the time the preservative is in contact with the protein, thus significantly reducing the associated stability risks. In the case of liquid formulations, the effectiveness and stability of the preservative should be maintained throughout the product's shelf life (approximately 18 to 24 months). It is important to note that the effectiveness of the preservative should be demonstrated in the final formulation containing the active pharmaceutical ingredient and all excipient components.

[0469] The antibody constructs disclosed herein can also be formulated as immunoliposomes. A “liposome” is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants, which can be used to deliver drugs to mammals. The components of a liposome are typically arranged in a bilayer, similar to the lipid arrangement of a biological membrane. Liposomes containing antibody constructs are prepared by methods known in the art, such as those 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. Patent Nos. 4,485,045 and 4,544,545; and WO 97 / 38731. Liposomes with extended circulation times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by a reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to produce liposomes of the desired diameter. The Fab' fragment of the antibody construct 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). Chemotherapy agents are optionally contained within the liposomes. See Gabizon et al., J. National Cancer Institute 81(19):1484 (1989).

[0470] Once formulated, a pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored in ready-to-use form or in a reconstituted form prior to application (e.g., lyophilized).

[0471] The bioactivity of the pharmaceutical compositions defined herein can be determined, for example, by cytotoxicity assays, as described in the following examples, WO 99 / 54440, or by Schlereth et al. (Cancer Immunol. Immunother. [Cancer Immunology Immunotherapy] 20 (2005), 1-12). As used herein, “efficacy” or “in vivo efficacy” means the response of a therapy to the pharmaceutical compositions of the present invention using, for example, standardized NCI response criteria. Success of a therapy using the pharmaceutical compositions of the present invention or in vivo efficacy refers to the effectiveness of the composition for its intended use, i.e., the composition’s ability to produce its desired effect, i.e., the ability to deplete pathological cells (e.g., tumor cells). In vivo efficacy can be monitored by established standard methods for the corresponding disease entity, including but not limited to white blood cell counts, differential cytometry, fluorescence-activated cell sorting, and bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used. In addition, computer-aided computed tomography (CT), X-ray, and magnetic resonance imaging (MRI) can be used (e.g., for 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-Hodgkin's lymphomas. [Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas] NCI-sponsored International Working Group. J Clin Oncol. [Journal of Clinical Oncology] April 1999; 17(4):1244), positron emission tomography, 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.

[0472] Another major challenge in developing drugs (such as the pharmaceutical compositions of the present invention) is the predictable modulation of pharmacokinetic properties. To this end, pharmacokinetic profiles of candidate drugs can be established, i.e., profiles of pharmacokinetic parameters affecting the ability of a particular drug to treat a given symptom. Pharmacokinetic parameters affecting the ability of a drug to treat a disease entity include, but are not limited to, half-life, distribution capacity, hepatic first-pass metabolism, and serum binding. The efficacy of a given agent can be affected by each of the parameters mentioned above. The contemplated features of the antibody constructs of the present invention provide for specific FC patterns they contain, such as differences in pharmacokinetic behavior. Targeting antibody constructs with extended half-life according to the present invention preferably exhibit a surprisingly increased in vivo retention time compared to the “canonical” non-HLE form of said antibody constructs. “Half-life” refers to the time it takes for 50% of the administered drug to be eliminated through biological processes (e.g., metabolism, excretion, etc.). “Hepatic first-pass metabolism” refers to the tendency of a drug to be metabolized upon its first contact with the liver, i.e., during its first passage through the liver. "Volume of distribution" refers to the degree to which a drug remains in various compartments of the body (such as intracellular and extracellular spaces, tissues, and organs), and the distribution of the drug within these compartments. "Serium binding" refers to the tendency of a drug to interact with and bind to serum proteins (such as albumin), thereby reducing or eliminating its biological activity.

[0473] Pharmacokinetic parameters also include bioavailability, lag time (T lag), Tmax, absorption rate, onset time, and / or Cmax for a given amount of drug administered. "Bioavailability" refers to the amount of drug in the blood compartment. "Lack time" refers to the time delay between drug administration and its detectability and measurability in blood or plasma. "Tmax" is the time after which the drug reaches its maximum blood concentration, and "Cmax" is the maximum blood concentration obtained with a given drug. The time required to reach the blood or tissue concentration of the drug required to achieve its biological effect is influenced by all parameters. Pharmacokinetic parameters of antibody constructs exhibiting cross-species specificity (which can be determined in preclinical animal testing in non-chimpanzee primates as outlined above) are also presented, for example, in the publications of Schlereth et al. (Cancer Immunol. Immunother. [Cancer Immunology Immunotherapy] 20 (2005), 1-12).

[0474] In a preferred aspect, the pharmaceutical composition is stable at about -20°C for at least four weeks. It is clear from the accompanying examples that different systems can be used to test the quality of the antibody constructs of the present invention against the quality of corresponding existing antibody constructs. These tests are understood to be consistent with “ICH Harmonised Tripartite Guideline: Stability Testing of Biotechnological / Biological Products Q5C and Specifications: Test procedures and Acceptance Criteria for Biotech Biotechnological / Biological Products Q6B” and are therefore selected to provide stability indicator curves that provide certainty in detecting changes in product identity, purity, and potency. The term purity is generally accepted as relative. Due to the effects of glycosylation, deamidation, or other heterogeneity, the absolute purity of biotechnological / biological products should typically be assessed by more than one method, and the derived purity value depends on the method. For the purposes of stability testing, purity testing should focus on the method used to identify degradation products.

[0475] To assess the quality of a pharmaceutical composition containing the antibody construct of the present invention, it can be analyzed, for example, by analyzing the content of soluble aggregates in solution (based on size exclusion HMWS). Preferably, stability at about -20°C for at least four weeks is characterized by a content of less than 1.5% HMWS, preferably less than 1% HMWS.

[0476] The formulations described herein are pharmaceutical compositions intended for use in patients in need of treating, improving, and / or preventing the pathological medical conditions described herein. The term "treatment" refers to both therapeutic and prophylactic (or preventative) measures. Treatment includes the application or administration of the formulation to the body, isolated tissues, or cells of a patient having a disease / disorder, symptoms of a disease / disorder, or a predisposition to a disease / disorder, with the aim of treating, curing, alleviating, reducing, altering, remedying, mitigating, improving, or influencing the disease, its symptoms, or the predisposition to the disease.

[0477] As used herein, the term “improvement” refers to any improvement in the disease status of a patient with the diseases specified below, achieved by administering an antibody construct to a subject in need. Such improvement may also be interpreted as a slowing or halting of disease progression in the patient. As used herein, the term “prevention” means the avoidance of the onset or recurrence of the disease in a patient with the tumors or cancers or metastatic cancers specified below, achieved by administering an antibody construct to a subject in need.

[0478] The term "disease" refers to any condition that would benefit from treatment with the antibody constructs or pharmaceutical compositions described herein. This includes chronic and acute disorders or diseases, including those pathological conditions that make mammals susceptible to the diseases discussed.

[0479] A “growth” is an abnormal growth of tissue that usually, but not always, forms a lump. When it does form a lump, it is usually called a “tumor.” A growth or tumor can be benign, potentially malignant (precancerous), or malignant. Malignant tumors are commonly referred to as cancer. They often invade and destroy surrounding tissues and can form metastases, meaning they spread to other parts of the body, tissues, or organs. Therefore, the term “metastatic cancer” covers metastases to tissues or organs other than the original tumor. Lymphoma and leukemia are lymphoid tumors. For the purposes of this invention, they are also covered in the terms “tumor” or “cancer.”

[0480] The terms "subjects in need" or "those in need of treatment" include those who already have a disorder as well as those whose disorder needs to be prevented. Subjects in need, or "patients," include people and other mammalian subjects who receive preventative or therapeutic treatment.

[0481] The antibody constructs of the present invention are typically designed for specific routes and methods of administration, specific dosages and frequencies of administration, specific treatments for specific diseases, bioavailability, and duration of action. The materials of the composition are preferably formulated at concentrations acceptable for the application site.

[0482] Therefore, formulations and compositions according to the present invention can be designed for delivery via any suitable route of administration. Routes of administration include, but are not limited to:

[0483] • Local routes (such as epidermal, inhalation, nose, eye, ear (auricular / aural), vagina, mucous membrane);

[0484] • Enteric routes (such as oral, gastrointestinal, sublingual, sublipal, buccal, rectal); and

[0485] • Extra-gastrointestinal routes (such as intravenous, intra-arterial, intra-bone, intramuscular, intracerebral, intravenous, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intra-articular, intracardiac, intradermal, intralesional, intrauterine, intrabladder, intravitreal, percutaneous, intranasal, transmucosal, intrasynovial, and intraluminal).

[0486] Pharmaceutical compositions and antibody constructs are particularly suitable for parenteral administration, such as subcutaneous or intravenous delivery, such as by injection (e.g., pellet injection) or by infusion (e.g., continuous infusion). Pharmaceutical compositions can be administered using medical devices. 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.

[0487] If the pharmaceutical composition has been lyophilized, the lyophilized material is first reconstituted in a suitable liquid prior to administration. The lyophilized substance can be reconstituted in, for example, antibacterial water for injection (BWFI), physiological saline, phosphate-buffered saline (PBS), or the same formulation in which the protein was previously in the lyophilized form.

[0488] The composition can be administered to a subject at an appropriate dose, which can be determined, for example, by dose-escalation studies of the antibody construct of the invention exhibiting the cross-species specificity described herein, administered in increasing doses to non-chimpanzee primates (e.g., rhesus monkeys). As described above, the antibody construct of the invention exhibiting the cross-species specificity described herein can advantageously be used in the same form for preclinical testing in non-chimpanzee primates and for use as a drug in humans. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors, including patient size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, and other concurrently administered medications.

[0489] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutic effective dose" is defined as an amount sufficient to cure or at least partially stop the disease and its complications in a patient with an existing condition. The effective amount or dosage for this purpose will depend on the condition to be treated (indication), the antibody construct delivered, the treatment context and objectives, the severity of the disease, prior therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's body size (weight, body surface or organ size) and / or condition (age and general health status), and the general state of the patient's autoimmune system. Appropriate dosages may be adjusted according to the attending physician's judgment so that they can be administered to the patient in a single dose or in a series of doses, with the aim of achieving optimal therapeutic effect. Depending on the above factors, typical dosage ranges can be from about 0.1 μg / kg to up to about 30 mg / kg or higher. In specific embodiments, the dosage range can be from 1.0 μg / kg to about 20 mg / kg, optionally from 10 μg / kg to up to about 10 mg / kg or from 100 μg / kg to up to about 5 mg / kg. Therapeutic effective doses of the antibody construct preferably result in a reduction in the severity of disease symptoms, an increase in the frequency or duration of symptom-free periods, or prevention of damage or disability due to disease suffering. For the treatment of diseases associated with MUC17 expression as described above, the therapeutically effective dose of the antibody construct of the present invention (here: anti-MUC17 / anti-CD3 antibody construct) preferably inhibits cell growth or tumor cell 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% relative to untreated patients. The ability of the compound to inhibit tumor growth can be evaluated in animal models where efficacy is predicted.

[0490] The pharmaceutical composition may be administered as the sole therapeutic agent or, as needed, in combination with other therapies such as anticancer therapies (e.g., other protein and non-protein drugs). These drugs may be administered concurrently with compositions containing antibody constructs as defined herein, or separately at time-defined intervals and doses before or after the administration of the antibody construct.

[0491] As used herein, the term "effective and non-toxic dose" refers to a tolerable dose of the antibody construct that is sufficiently high to induce pathological cell depletion, tumor elimination, tumor shrinkage, or disease stabilization without or substantially without major toxic effects. Such an effective and non-toxic dose can be determined, for example, through dose-escalation studies as described in the art, and this effective and non-toxic dose should be below the dose that induces serious adverse events (dose-limiting toxicity, DLT). As used herein, the term "toxicity" refers to the toxic effects of a drug manifested in adverse events or serious adverse events. These side effects may refer to a lack of systemic tolerance and / or a lack of local tolerance to the drug after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0492] As used herein, the terms “safety,” “in vivo safety,” or “tolerability” define the administration of a drug without immediately inducing serious adverse events (local tolerance) or without inducing serious adverse events during a longer period of drug administration. For example, “safety,”...

Claims

1. An antibody that binds to human MUC17 as shown in SEQ ID NO: 1, wherein the antibody binds to cell surface-associated MUC17 protein, wherein the antibody comprises a variable heavy chain and a variable light chain, the variable heavy chain containing heavy chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NO: 2, 3 and 4, and the variable light chain containing light chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NO: 6, 7 and 8.

2. The antibody according to claim 1, wherein the antibody comprises a VH region as shown in SEQ ID No: 5 and / or a VL region as shown in SEQ ID No:

9.

3. A polynucleotide encoding the antibody according to claim 1.

4. A host cell, wherein the host cell has been transformed or transfected with the polynucleotide according to claim 3.

5. A composition comprising the antibody according to claim 1.

6. A detection system comprising the antibody according to claim 1 or the composition according to claim 5.

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