CDH17 antibodies and methods of treating cancer

Abstract

The present disclosure provides antigen binding proteins that bind to CDH17; a bispecific antigen binding protein that binds to CDH17 and a second antigen; and conjugates thereof. Also provided herein are related polypeptides, nucleic acids, vectors, host cells, and conjugates. In addition, kits and pharmaceutical compositions comprising these entities are also provided. Methods of making the antigen binding proteins and methods of treating subjects having cancer are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 286,894, filed on December 7, 2021, and U.S. Provisional Application No. 63 / 349,258, filed on June 6, 2022, the entire contents of each of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present disclosure generally relates to antibodies specific for E-cadherin-17 (CDH17) and their use in treating cancer. Background Art

[0004] Antibodies are powerful therapeutic agents characterized by their ability to specifically target different antigens on cells, bacteria, viruses or toxins with limited side effects. To meet the medical needs of patients with CDH17-related diseases, it is clinically necessary to provide new antibodies, such as the antibodies described herein. Summary of the invention

[0005] Provided herein are antigen binding proteins that bind to CDH17. In various aspects, the antigen binding proteins of the present disclosure bind to human CDH17 and optionally bind to mouse CDH17. In various aspects, the antigen binding protein binds to the extracellular domain (ECD) of CDH17. In various embodiments, the present disclosure provides an antigen binding protein for CDH17.

[0006] In various cases, the antigen binding protein binds to CDH17 and does not bind to any other member of the CDH family. In various aspects, the antigen binding protein binds to CDH17 endogenously expressed by human cancer cells, such as HPAF-II pancreatic cancer cells. In various cases, the antigen binding protein of the present disclosure inhibits tumor growth in a subject (e.g., a human) without any other moiety attached to the antigen binding protein. In various cases, an antigen binding protein that is not conjugated to a heterologous moiety (e.g., not conjugated to any chemotherapeutic agent, drug, or toxic moiety) inhibits tumor growth in a subject (e.g., a human).

[0007] In various aspects, the antigen binding protein binds to CDH17 expressed by human cancer cells. In various aspects, the antigen binding protein inhibits the binding interaction between human CDH17 and the reference anti-CDH17 antibody. Without being bound by a particular theory, the inhibitory effect of the antigen binding protein provided herein allows such entities to be used in methods for reducing tumor growth and treating subjects with tumors or cancer. As further discussed herein, in various aspects, the antigen binding protein is an antibody, an antibody fragment thereof that binds an antigen, or an antibody protein product.

[0008] The present disclosure also provides antigen binding proteins comprising at least 3, 4, 5 or all amino acid sequences of a set of specific amino acid sequences. In various aspects, the antigen binding protein comprises at least 3, 4, 5 or 6 complementary determining region (CDR) amino acid sequences of the CDH17 antibody disclosed herein.

[0009] The present disclosure also provides antigen binding proteins comprising the amino acid sequences detailed herein.

[0010] The present disclosure provides a bispecific antigen binding protein that binds to CDH17 and a second antigen. The bispecific antigen binding protein may comprise any of the antigen binding proteins described herein. The second antigen may be a cell surface protein, optionally a protein that binds to regulate an immune response. The bispecific antigen binding protein may adopt any structure, such as a diabody, TandAb (tandem diabody), BiTE (bispecific T cell adapter), etc.

[0011] The present disclosure also provides a conjugate comprising an antigen binding protein or a bispecific antigen binding protein and a heterologous portion (e.g., a cytotoxic drug). The conjugate may comprise a cleavable linker or a non-cleavable linker. The conjugate may have a different number of heterologous portions (agents) conjugated to an antigen binding protein or a bispecific antigen binding protein as described herein, preferably 1-8 agents per protein or 3-8 agents per protein. The conjugate may be a site-specific conjugate. The conjugate may be a homogeneous conjugate or a heterogeneous conjugate.

[0012] Related polypeptides, nucleic acids, vectors, host cells and conjugates are also provided herein. In addition, kits and pharmaceutical compositions comprising these entities are also encompassed.

[0013] Also provided are methods of preparing antigen binding proteins. In various embodiments, the methods include culturing a host cell to express an antigen binding protein or polypeptide, the host cell comprising a nucleic acid encoding an antigen binding protein or polypeptide described herein.

[0014] Additionally provided herein are methods of treating a subject having cancer. In various embodiments, the method comprises administering to the subject an amount of a pharmaceutical composition of the present disclosure effective to treat the subject's cancer.

[0015] Also provided are methods of treating a subject having a cancer that expresses CDH17, the method comprising administering to the subject a pharmaceutical composition described herein. In various embodiments, the cancer that expresses CDH17 expresses CDH17. Also contemplated is a method of inhibiting tumor growth in a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0016] A method of reducing tumor size or preventing recurrence of cancer in a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0017] Also provided herein is a method of treating cancer in a subject diagnosed as an overexpresser of CDH17, the method comprising administering the pharmaceutical composition described herein to the subject.

[0018] In various embodiments, the administration induces apoptosis of tumor cells, such as cells expressing CDH17, thereby causing tumor regression or slowing tumor growth. In various embodiments, the administration induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or clearance, and / or disruption of tumor cell adhesion, each of which causes tumor regression or slowing tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Graph representing CDH17 expression in human cancers.

[0020] FIG. 2A to FIG. 2B The sequence comparison results of human CDH17 (UniProtKB accession number Q12864) with cynomolgus macaque CDH17 (UniProtKB accession number A0A2K5X8I8), mouse CDH17 (UniProtKB accession number Q9R100) and rat CDH17 (UniProtKB accession number P55281) expressed as percentage of identity, as well as the phylogenetic diagram and topological structure of CDH17 transmembrane proteins (A) and aligned sequences, in which the yellow highlight represents the extracellular domain of human CDH17 (B).

[0021] FIG3 shows the tumor volume (mm) of mice bearing human CDH17-positive colorectal tumors (SNU-C1; ATCC CRL-5972) after weekly treatment with 10 mg / kg of control IgG antibody (human IgG1) or 6 different chimeric monoclonal antibodies against CDH17 protein. 3 ) with time (days) (A), mean change in tumor volume on day 32 (mm 3 )(B) and percent change in body weight (C). The chimeric monoclonal antibody comprises mouse VH and VL sequences, human IgG1 constant region and human Igκ constant region.

[0022] FIG4 shows tumor volume (mm) of mice bearing human CDH17-positive colorectal tumors (SNU-C1; ATCC CRL-5972) after weekly treatment with 10 mg / kg control human IgG1 antibody, two different chimeric monoclonal anti-CDH17 antibodies (CDH17-653-m or CDH17-657-m), or eight different humanized anti-CDH17 antibodies (CDH17-646-h7, CDH17-653-h42, CDH17-653-h43, CDH17-657-h16, CDH17-663-h7, CDH17-670-h12, CDH17-675-h11, or CDH17-683-h6). 3 ) changes over time (days) (A), tumor volume on day 28 (mm 3 ) and percent body weight change (C); note that "CDH17-" can be replaced with "07-0" to refer to the same antibody (e.g., the antibody designated as CDH17-646-h7 is the same antibody as the antibody designated as 07-0646-h7; CDH17-663-h7 is also known as 07-0663-h7 and CDH17hAb0663-h7).

[0023] Figure 5 shows the tumor volume (mm) of mice bearing human CDH17 positive pancreatic tumors (HPAF-II) after weekly treatment with 10 mg / kg control human IgG1 antibody or eight different humanized anti-CDH17 antibodies (CDH17-646-h7, CDH17-653-h42, CDH17-653-h43, CDH17-657-h16, CDH17-663-h7, CDH17-670-h12, CDH17-675-h11 or CDH17-683-h6). 3 ) changes over time (days) (A) and tumor volume (mm 3 ) of the average change (B).

[0024] Figure 6 shows the tumor volume (mm) of mice bearing the human CDH17-negative melanoma cell line (M202) after weekly treatment with 10 mg / kg control human IgG1 antibody or six different humanized anti-CDH17 antibodies (CDH17-646-h7, CDH17-653-h42, CDH17-653-h43, CDH17-663-h7, CDH17-670-h12 or CDH17-683-h6). 3 ) over time (days) (A) and tumor volume (mm 3 ) Plot of the average change (B).

[0025] Figure 7 shows a comparison of the selective tumor growth inhibition induced by the antibody-drug conjugate (ADC; VC-PAB-MMAE) of the chimeric monoclonal anti-CDH17 antibody CDH17-653 with unconjugated antibody or control IgG antibody in mice bearing human CDH17 positive tumors (AC) and human CDH17 negative tumors (DF). Xenografts were obtained from human colorectal cancer cell line SNU-C1 (A), human pancreatic cancer cell line PaTu8988s (B), human colorectal cancer cell line LS513 (C), human small cell lung cancer cell lines H524 (D) and COR-L279 (E), and human melanoma cell line M202 (F). Mice were treated with 5 mg / kg ADC or 10 mg / kg unconjugated parental CDH17-653 or IgG control by IV tail vein injection, with repeated doses 3 times per week.

[0026] FIG8 shows the increase in tumor volume (mm) induced by three different humanized anti-CDH17 antibody-drug conjugates (ADC; VC-PAB-MMAE) in CDH17-positive human colorectal cancer xenografts (SNU-C1) after treatment with 3 repeated doses of 5 mg / kg ADC per week. 3 ) Tumor growth inhibition over time (A), tumor volume on day 42 (mm 3 ) Mean change (B) and percent change in body weight (C). Antibody-drug conjugates: CDH17-ADC-646-h7, CDH17-ADC-653-h43, and CDH17-ADC-663-h7. IgG is a non-targeting control antibody.

[0027] FIG9 shows the increase in tumor volume (mm) induced by three different humanized anti-CDH17 antibody-drug conjugates (ADC; VC-PAB-MMAE) in CDH17-positive human pancreatic cancer xenografts (HPAF-II) after treatment with 3 repeated doses of 5 mg / kg ADC per week. 3 ) Tumor growth inhibition over time (A) or tumor volume on day 27 (mm 3 ) (B). Antibody-drug conjugates: CDH17-ADC-646-h7, CDH17-ADC-653-h43, and CDH17-ADC-663-h7. IgG is a non-targeting control antibody.

[0028] Figure 10 shows the tumor volume (mm) of mice bearing human CDH17 negative melanoma (M202) after treatment with 3 repeated doses of 5 mg / kg control IgG antibody or humanized anti-CDH17 antibody-drug conjugate (VC-PAB-MMAE) (CDH17-ADC-646-h7, CDH17-ADC-653-h43 or CDH17-ADC-663-h7) per week. 3 ) over time (days) (A) or tumor volume (mm 3 ) (B) shows the loss of antitumor activity of the ADC in CDH17-negative xenografts.

[0029] Fig.11 Shown is the time course of internalization of humanized anti-CDH17 antibodies 07-0646-h7 (upper panel) and 07-0663-h7 (lower panel) in the CDH17-positive human colorectal cancer cell line LS513.

[0030] Fig.12 Shown is the time course of internalization of humanized anti-CDH17 antibodies 07-0646-h7 (upper panel), 07-0653-h33 (middle panel), and 07-0663-h7 (lower panel) in the CDH17-positive human pancreatic cancer cell line HPAF-II.

[0031] Fig.13 Shown is the time course of internalization of humanized anti-CDH17 antibody-drug conjugates (ADCs) 07-0646-h7-VC-PAB-MMAE (upper panel) and 07-0653-h43-VC-PAB-MMAE (lower panel) in the CDH17-positive human colorectal cancer cell line LS513.

[0032] Fig.14 Shown is the time course of internalization of a humanized anti-CDH17 antibody (07-0663-h7) conjugated to: monomethyl auristatin E, MMAE (07-0663-h7-VC-PA B-MMAE; top panel); or Alexa Fluor 647, AF647 (07-0663-h7-AF647; bottom panel) in the CDH17-positive human colorectal cancer cell line LS513.

[0033] Fig.15Flow cytometry is shown to evaluate the binding activity of three humanized anti-CDH17 antibodies to HEK293 cells overexpressing human, monkey, mouse or rat CDH17 as fusion proteins with mGFP (human or mouse CDH17) or moxGFP (monkey or rat CDH17) fluorescent proteins. Upper panel - anti-CDH17 signal after detection of anti-CDH17 antibody using fluorescently labeled secondary antibody; lower panel - GFP signal.

[0034] Fig.16 The dissociation constants K determined for the binding of three humanized anti-CDH17 antibodies to HEK293T cells are summarized. D , the cells overexpress a fusion protein comprising human CDH17-mGFP, monkey CDH17-moxGFP, mouse CDH17-mGFP or rat CDH17-moxGFP protein.

[0035] Fig.17 The biochemical, biophysical, and cell biological characteristics of the three best humanized anti-CDH17 antibodies and properties of some commercial antibodies are summarized.

[0036] Fig.18 Shown is SDS-PAGE analysis of the stability of three humanized anti-CDH7 antibodies stored at -80°C, 4°C, room temperature or 37°C for 5 weeks, wherein the left side of each gel shows antibodies stored under reducing conditions and the right side shows antibodies stored under non-reducing conditions immediately before electrophoresis.

[0037] Fig.19 Shown are size exclusion chromatograms and SDS-PAGE analysis of humanized anti-CDH7 antibody 07-0646-h7 before and after treatment with peptide-N-glycosidase F (PNGase F).

[0038] Fig. 20 Results of epitope mapping of CDH17 hAb 07-0663-h7 binding to peptides spanning the ECD of CDH17 are shown. CDH17 hAb 07-0663-h7 binds to 3 peptides with overlapping 7 amino acid regions, LDANGII. Mutations were introduced in the 7 amino acid core region to further refine the CDH17 hAb 07-0663-h7 binding region.

[0039] Fig.21 Epitope mapping data of CDH17 hAb 07-0663-h7 are shown, indicating that the 5-amino acid sequence DANGI is the key epitope core region bound by CDH17 hAb 07-0663-h7.

[0040] Figure 22 shows two graphs showing (A) ADCC efficacy of 07-0646-h7 in LS513, SNUC1 and M202 cells, and (B) ADCC efficacy of various CDH17 antibodies in LS513 cells. NFAT activation was assessed by determining Lucia luciferase activity in the supernatant, which indicated that ADCC reactions were induced. The data show that CDH17 antibodies, particularly 07-646-h7 and 07-663-h7, induce ADCC in CDH17 positive cell lines.

[0041] Fig.23 CDH17-CD3 bispecific antibodies and their effects on T cell activation are shown. Treatment of CDH17-positive cell lines (HPAF2 and SNUC1) with the 07-0653-h43Bs bispecific antibody caused T cell activation compared to the no-target cell control. In contrast, for the 07-0646-h7Bs and 07-0663-h7Bs bispecific antibodies, there was no difference in the level of T cell activation between CDH17-positive cells and the no-target cell control. DETAILED DESCRIPTION

[0042] The present disclosure describes antigen binding proteins directed against CDH17, such as those specific for CDH17, for use in treating cancers expressing CDH17.

[0043] Antigen Binding Protein

[0044] Provided herein is an antigen binding protein that binds to CDH17. In various embodiments, the antigen binding protein binds to an isotype of CDH17. The antigen binding protein of the present disclosure may be in any of the various forms of antigen binding proteins known in the art. In various embodiments, the antigen binding protein of the present disclosure is in the form of an antibody, or an antibody fragment that binds to an antigen, or an antibody protein product.

[0045] In various embodiments of the present disclosure, the antigen-binding protein comprises, is essentially composed of, or is composed of an antibody. As used herein, the term "antibody" refers to a protein in the form of a conventional immunoglobulin, which comprises a heavy chain and a light chain, and comprises a variable region and a constant region. For example, an antibody can be an IgG, which is a "Y-shaped" structure formed by two pairs of identical polypeptide chains, each pair having a "light" chain (usually having a molecular weight of about 25 kDa) and a "heavy" chain (usually having a molecular weight of about 50-70 kDa). The antibody has a variable region and a constant region. In the IgG form, the variable region is generally about 100-110 or more amino acids, comprises three complementary determining regions (CDRs), is primarily responsible for antigen recognition, and is very different between other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is composed of the N-terminal region of each light chain and heavy chain, while the constant region is composed of the C-terminal portion of each heavy chain and light chain. (Janeway et al., "Structure of the Antibody Molecule and the ImmunoglobulinGenes", Immunobiology: The Immune System in Health and Disease, 4th Edition, ElsevierScience Ltd. / Garland Publishing, (1999)).

[0046] The general structure and characteristics of antibody CDR have been described in the art. In short, in antibody scaffolds, CDR is embedded in the framework of heavy chain and light chain variable region, in which they constitute the region mainly responsible for antigen binding and recognition. The variable region is generally included in the framework region (designated as framework region 1-4, i.e., FR1, FR2, FR3 and FR4, according to Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; also see Chothia and Lesk, 1987, J.Mol.Biol.196:901-917) at least three heavy chains or light chain CDRs (Kabat et al., 1991; also see Chothia and Lesk, 1987, the same; Chothia et al., 1989, Nature 342:877-883). CDR can be annotated in a variety of ways, including according to the method annotations of Kabat, AbM or IMGT. Therefore, depending on which method is used to annotate the CDR sequences, the CDRs of the same antibody may contain different sequences. This is exemplified in the table provided herein.

[0047] In a related embodiment, the residues of the framework are changed. The heavy chain framework region that can be changed is located in the region designated as H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chain CDR residues, and the light chain framework region residues that can be changed are located in the region designated as L-FR1, L-FR2, L-FR3 and L-FR4, which surround the light chain CDR residues. The amino acid in the framework region can be replaced by any suitable amino acid identified, for example, in a human framework or a human consensus framework.

[0048] The antibody may comprise any constant region known in the art. Human light chains are classified as kappa light chains and lambda light chains. Heavy chains are classified as μ, δ, γ, α or ε, and the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. The embodiments disclosed herein include all of these antibody classes or isotypes. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an α type, a δ type, an ε type, a γ type or a μ type heavy chain constant region, such as a human α type, a δ type, an ε type, a γ type or a μ type heavy chain constant region. Therefore, in various embodiments, the antibody is an antibody of the following isotypes: IgA, IgD, IgE, IgG or IgM, including any one of IgG1, IgG2, IgG3 or IgG4. In various aspects, to improve half-life / stability or to make the antibody more suitable for expression / manufacturability, the antibody comprises a constant region comprising one or more amino acid modifications relative to the naturally occurring counterpart. In various cases, the antibody comprises a constant region in which the C-terminal Lys residue present in the naturally occurring counterpart is removed or cleaved.

[0049] The antibody can be a monoclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, or human. In this regard, the antibody can be considered to be a mammal antibody, such as a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a human antibody, or the like. In some aspects, the antigen-binding protein is an antibody, such as a human antibody. In some aspects, the antigen-binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody containing a domain from two or more different antibodies. A chimeric antibody can, for example, contain a constant domain from one species and a variable domain from another species, or more generally, can contain an amino acid sequence segment from at least two species. A chimeric antibody can also contain a domain from two or more different antibodies in the same species. When used with respect to an antibody, the term "humanization" refers to an antibody having at least a CDR region from a non-human source, and the CDR region is engineered to have a structure and immune function that is more similar to a true human antibody than the original source antibody. For example, humanization can involve the CDR of non-human antibodies such as mouse antibodies being transplanted into human antibodies. Humanization can also involve selecting amino acid substitutions to make non-human sequences more similar to human sequences. Information can be publicly obtained through other well-known databases in the Uniprot database and antibody engineering and production fields, including sequence information of human antibody heavy chain and light chain constant regions. For example, IgG1 constant region can be obtained from the Uniprot database, as described below, and the database is incorporated herein by reference. In addition, in another example, IgG2 constant region can be obtained from the Uniprot database with Uniprot numbering P01859, which is incorporated herein by reference.

[0050] By way of example only, the sequence of a murine immunoglobulin kappa light chain constant region or an immunoglobulin gamma-2A heavy chain constant region includes the following.

[0051]

[0052]

[0053] By way of example only, the sequences of a human immunoglobulin kappa light chain constant region, a human immunoglobulin lambda light chain constant region 2, a human IgG1 heavy chain constant region, and a human IgG2 heavy chain constant region include the following sequences.

[0054]

[0055]

[0056] In some embodiments, when the light chain variable region (VL sequence) and light chain framework 4 (LFR4) are identified according to the Kabat and AbM methods, the VL sequence and LFR4 can be identified as ending with the tripeptide RTV at its C-terminus (see, for example, the amino acid sequences shown in 07-0646-h7 (Table 2B and Table 2C), 07-0653-h43 (Table 3B and Table 3C), and 07-0663-h7 (Table 4B and Table 4C). In the case where the VL sequence and LFR4 end with the RTV tripeptide, the light chain constant region that is joined to the VL sequence to form a complete immunoglobulin κ light chain (without its signal sequence) is an immunoglobulin κ light chain constant region having a sequence as given in UniProt ID: P01834 but lacking RTV at the amino terminus. This avoids the repetition of the RTV tripeptide sequence in the fully reassembled immunoglobulin κ light chain. In some embodiments, the substituted human immunoglobulin κ constant region light chain comprises the following amino acid sequence:

[0057] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.

[0058] In other embodiments, when the amino acid sequences of the immunoglobulin light chain variable region (VL sequence) and the light chain framework 4 (LFR4) are defined using the IMGT method, these sequences do not end with the RTV tripeptide sequence, but end with the sequence LEIK at their C-terminus (see, for example, the amino acid sequences shown in 07-0646-h7 (Table 2A), 07-0653-h43 (Table 3A), and 07-0663-h7 (Table 4A). In some such embodiments, the immunoglobulin kappa light chain constant region that is joined to the VL to produce a complete human kappa light chain (without a signal sequence) is the complete amino acid sequence provided by UniProt ID: P01834.

[0059] In some embodiments, the nucleic acid sequence encoding a complete immunoglobulin kappa light chain (without its signal sequence) avoids repetition of the RTV tripeptide sequence found at the border of the light chain variable region (as shown in Table 2B, Table 2C, Table 3B, Table 3C, Table 4B and Table 4C) and the constant region (UniProt ID: P01834).

[0060] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to produce two Fab fragments and one Fc fragment. Pepsin cleaves antibodies to produce F(ab')2 fragments and pFc' fragments. In various aspects of the present disclosure, the antigen-binding proteins of the present disclosure are antigen-binding fragments of antibodies (also referred to as antigen-binding antibody fragments, antigen-binding fragments, antigen-binding portions). In various cases, the antigen-binding antibody fragment is a Fab fragment or a F(ab')2 fragment.

[0061] The structure of antibodies has been used to create more and more alternative antibody forms, with molecular weights ranging from at least about 12-150 kDa, and valences (n) ranging from monomers (n=1) to dimers (n=2), to trimers (n=3), to tetramers (n=4) and even higher; these alternative antibody forms are referred to herein as "antibody protein products". Antibody protein products include products based on the structure of complete antibodies and products that mimic antibody fragments that retain complete antigen binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment that retains a complete antigen binding site is the Fv fragment, which consists entirely of the variable (V) region. A soluble, flexible amino acid peptide linker is used to connect the V region to the scFv (single-chain fragment variable) fragment to stabilize the molecule, or a constant (C) domain is added to the V region to produce a Fab fragment [fragment, antigen binding]. Both scFv and Fab fragments can be easily produced in host cells, such as prokaryotic host cells. Other antibody protein products include scFv stabilized by disulfide bonds (ds-scFv); single-chain Fab (scFab); and dimer and multimer antibody forms, such as diabodies, triabodies and tetrabodies; or miniantibodies (miniAbs) in different forms consisting of scFv connected to oligomerization domains. The smallest fragments are VHH / VH of camel heavy chain antibodies, and single domain antibodies (sdAbs). The building blocks most commonly used to create new antibody formats are single-chain variable (V) domain antibody fragments (scFvs), which contain V domains (VH and VL domains) from heavy and light chains connected by a peptide linker of about 15 amino acid residues. In some embodiments, the scFv can be an anti-CDH17 scFv comprising the light chain and heavy chain variable regions of any antibody currently described herein. In some embodiments, the scFv may be an anti-CDH17 scFv comprising the light chain and heavy chain variable regions of 07-0646-h7, 07-0653-h43 or 07-0663-h7 anti-CDH17 antibodies. In some embodiments, the scFv comprises the sequence of 07-0646-7scFv (SEQ ID NO: 96), 07-0653-h43scfv (SEQ ID NO: 97) or 07-0663-h7scfv (SEQ ID NO: 98). In some embodiments, 07-0646-7scFv (SEQ ID NO: 96), 07-0653-h43scfv (SEQ ID NO: 97) or 07-0663-h7scfv (SEQ ID NO: 98) scFv may be encoded by the nucleic acid sequence of SEQ ID NO: 102, 103 or 104, respectively. In some embodiments, any antigen binding protein of the present disclosure may further comprise a detectable label, which may facilitate detection and / or purification of the antigen binding protein.Thus, in some embodiments, 07-0646-7scFv (SEQ ID NO: 96), 07-0653-h43scfv (SEQ ID NO: 97) or 07-0663-h7scfv (SEQ ID NO: 98) scFv may also comprise a detectable marker. In some such embodiments, the detectable marker may be an epitope tag. As used herein, in some embodiments, the epitope tag may be a C-terminal (HIS)6 epitope tag. Peptibodies or peptide-Fc fusions are another type of antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are described in detail in the art. See, for example, Shimamoto et al., mAbs 4(5): 586-591 (2012).

[0062] Other antibody protein products include single chain antibodies (SCAs); diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five categories: BsIgG, bispecific and multispecific antibodies attached to IgG forms (appended IgG), bispecific antibody (BsAb) fragments, bispecific fusion proteins and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67 (2) Part A: 97-106 (2015).

[0063] In various aspects, the antigen-binding proteins of the present disclosure comprise any of these antibody protein products, consist essentially of any of these antibody protein products, or consist of any of these antibody protein products. In various aspects, the antigen-binding proteins of the present disclosure comprise any of the following, consist essentially of any of the following, or consist of any of the following: scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., double antibodies, three antibodies, four antibodies), miniAb, camelid heavy chain antibody peptide body VHH / VH, sdAb, double antibodies; three antibodies; four antibodies; bispecific or trispecific antibodies, BsIgG, bispecific and multispecific antibodies attached to IgG forms, BsAb fragments, bispecific fusion proteins and BsAb conjugates.

[0064] In various cases, the antigen binding proteins of the present disclosure are antibody protein products in monomeric form or in polymeric, oligomeric or multimeric form. In certain embodiments in which the antibody comprises two or more different antigen binding region fragments, the antibody is considered to be bispecific, trispecific or multispecific, or bivalent, trivalent or multivalent, depending on the number of different epitopes recognized and bound by the antibody.

[0065] In various embodiments, the anti-CDH17 antibody or antibody variant thereof is selected from the group consisting of: a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0066] In various aspects, the antigen binding proteins of the present disclosure are linked to therapeutic agents. As described below, the therapeutic agent can be any therapeutic agent known in the art, including but not limited to chemotherapeutic agents, cytokines and growth factors, cytotoxic agents, etc. See "Conjugates" below.

[0067] In various aspects, any polypeptide of the present disclosure, such as antigen binding protein, may also include a heterologous peptide or polypeptide. In some embodiments, the heterologous peptide or polypeptide may be a detectable marker or a label that can be directly detected (e.g., GFP) or an indirect detection label (e.g., using a secondary antibody bound to a label). Examples of detectable markers include various enzymes, prosthetic groups, and labels (e.g., histidine tags, myc tags, flag tags, etc.). Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of bioluminescent materials include luciferase, luciferin, fluorescent proteins (e.g., GFP, RFP, etc.) and aequorin.

[0068] CDH17 and epitopes

[0069] The antigen binding proteins of the present disclosure bind to CDH17. In some embodiments, CDH17 is a human CDH17 comprising the following amino acid sequence:

[0070] (SEQ ID NO:19).

[0071] In various aspects, the antigen binding proteins of the present disclosure bind to epitopes within the amino acid sequence of CDH17. In various aspects, CDH17 is human CDH17, and the antigen binding proteins of the present disclosure bind to epitopes within the amino acid sequence of human CDH17 (e.g., SEQ ID NO: 19). "Epitope" refers to CDH17 or a region thereof to which an antigen binding protein binds. In some embodiments, the epitope is a linear epitope. "Linear epitope" refers to a region of CDH17 or a region thereof to which an antigen binding protein binds, and the region is composed of continuous amino acids of the amino acid sequence of CDH17. The amino acids of a linear epitope are adjacent to each other in the primary structure of CDH17. Therefore, a linear epitope is a fragment or a portion of the amino acid sequence of an antigen (i.e., CDH17). In various other embodiments, the epitope is a conformational or structural epitope. "Conformational epitope" or "structural epitope" refers to an epitope composed of amino acids that are positioned adjacent to each other only when CDH17 is in its properly folded state. Unlike linear epitopes, the amino acids of conformational or structural epitopes are not adjacent to each other in the primary structure (ie, amino acid sequence) of CDH17. Conformational or structural epitopes are not composed of consecutive amino acids of the amino acid sequence of the antigen (CDH17).

[0072] In various aspects, the epitope is located within the extracellular domain (ECD) of CDH17 (eg, human CDH17). In various aspects, the epitope bound by the antigen binding protein is located within SEQ ID NO:51.

[0073] In various aspects, the antigen binding protein binds to human CDH17 and / or non-human CDH17. In various cases, the non-human CDH17 is CDH17 of chimpanzee, rhesus monkey, dog, cow, mouse, rat, zebrafish or frog. In various cases, the antigen binding protein binds to human CDH17 and / or mouse CDH17.

[0074] Afucosylated Antibodies

[0075] Many secreted proteins undergo post-translational glycosylation, a process in which sugar moieties (e.g., glycans, carbohydrates) are covalently attached to specific amino acids of a protein. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is attached to an asparagine at the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid except proline, and (2) O-linked glycosylation, in which the glycan is attached to a serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is slight heterogeneity in protein glycoforms due to the wide range of glycan structures associated with each site (O or N).

[0076] All N-glycans have a common core sugar sequence: Manα1–6(Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn-X-Ser / Thr(Man3GlcNAc2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) type, which consists of two N-acetylglucosamine (GalNAc) moieties and a large number (e.g., 5, 6, 7, 8, or 9) of mannose (Man) residues; (B) complex type, which contains more than two GlcNAc moieties and any number of other sugar types; or (C) hybrid type, which contains Man residues on one side of the branch and GlcNAc at the base of the complex branch.

[0077] N-linked glycans typically contain one or more monosaccharides, such as galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (ClcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N0 acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynonanoic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (Gal A), and mannuronic acid (Man A).

[0078] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex set of reactions results in the connection of a core glycan structure consisting essentially of two GlcNAc residues and three Man residues. The glycan complex formed in the endoplasmic reticulum is modified by the action of enzymes in the Golgi apparatus. If the sugar is relatively inaccessible to the enzyme, it will usually remain in its original HM form. If the enzyme is able to access the sugar, many Man residues will be cleaved off and the sugar will be further modified, resulting in a complex N-glycan structure. For example, mannosidase-1 located in the cis-Golgi of the Golgi apparatus can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8 located in the medial-Golgi of the Golgi apparatus fucosylates the glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0079] Therefore, the sugar composition and structural configuration of the glycan structures will vary depending on the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the machinery enzymes to the glycan structures, the order of action of each enzyme, and the stage of protein release from the glycosylation machinery.

[0080] In an exemplary embodiment of the present disclosure, the antigen binding protein comprises an Fc polypeptide. As used herein, the term "Fc polypeptide" includes polypeptides derived from the native form and mutant form of the Fc region of an antibody. In exemplary aspects, the Fc polypeptide of the antigen binding protein currently disclosed comprises a glycan. In various cases, the glycan lacks fucose or is afucosylated. In exemplary aspects, the antigen binding protein comprises afucosylated glycan. As used herein, the term "afucosylated glycan" or "afucosylated glycoform" or "Afuc" refers to a glycoform lacking core fucose, such as an α1,6-linked fucose on a GlcNAc residue involving an amide bond to the Asn of the N-glycosylation site. Afucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Other non-fucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0081] The present disclosure also provides a composition, such as a pharmaceutical composition, comprising an antigen binding protein comprising an Fc polypeptide containing non-fucosylated glycans. In exemplary aspects, at least or about 25% of the antigen binding proteins present in the composition are antigen binding proteins comprising an Fc polypeptide containing non-fucosylated glycans. In exemplary aspects, at least or about 25% of the antigen binding proteins present in the composition are non-fucosylated. Optionally, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the antigen binding proteins present in the composition are non-fucosylated. Methods for producing compositions comprising antigen binding proteins of specific sugar profiles are known in the art. In exemplary embodiments, the antigen binding proteins are recombinantly produced in cells that have been genetically modified to alter the activity of enzymes in the de novo synthesis pathway or the salvage pathway. These two fucose metabolic pathways are shown in Figure 29B. In an exemplary embodiment, the cell is genetically modified to alter the activity of any one or more of the following: fucosyltransferase (FUT, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucokinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the cell is genetically modified to knock out a gene encoding FX. See, e.g., International Patent Publication No. WO2017 / 079165A1; Kanda et al., J Biotechnol 130, 2007, 300-310; Yamane-Ohunuki et al., Biotechnol Bioeng 87, 2004, 614-622; Malphettes et al., Biotechnol Bioeng 106, 2010, 774-783.

[0082] Bispecific formats

[0083] In exemplary aspects, the antigen binding protein is bispecific and is thus capable of binding to two different and distinct antigens.In exemplary embodiments, the antigen binding protein is bispecific and binds to CDH17 and a second antigen.

[0084] In an exemplary case, the second antigen is a cell surface protein expressed by T cells. In an exemplary aspect, the cell surface protein is a component of a T cell receptor (TCR), such as CD3. In an exemplary case, the second antigen is a co-stimulatory molecule that assists T cell activation, such as CD40 or 4-1BB (CD137). In an exemplary aspect, the second antigen is an Fc receptor. In various aspects, Fc receptors are Fcγ receptors, Fc-α receptors, and Fc-ε receptors. In an exemplary aspect, Fc receptors are CD64 (Fc-γRI), CD32 (Fc-γRIIA), CD16A (Fc-γRIIIA), CD16b (Fc-γRIIIb), FcεRI, CD23 (Fc-εRII), CD89 (Fc-εRI), Fcα / μR or FcRn. In an exemplary aspect, the Fc receptor is CD16A. In an exemplary case, the second antigen is an immune checkpoint molecule, such as a protein involved in an immune checkpoint path. Immune checkpoint paths and molecules or proteins that work therein are known in the art. See, e.g., Pardoll, Nat Rev Genet 12(4):252-264(2012). In exemplary cases, the immune checkpoint molecule is A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA or SIGLEC7. Optionally, the immune checkpoint molecule is PD-1, LAG3, TIM3 or CTLA4.

[0085] More than fifty forms of bispecific antigen-binding proteins are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7):838-847 (2015); Zhang et al., Exp Hematol Oncol 6:12 (2017); Spiess et al., Mol Immunol.; 67(2Pt A):95-106 (2015). In exemplary aspects, the bispecific antigen-binding proteins of the present disclosure are prepared by chemical engineering, genetic engineering or quadroma technology.

[0086] In exemplary aspects, bispecific antigen binding proteins are constructed from some or all of the constant domains of antibodies. In exemplary aspects, the bispecific antigen binding proteins of the present disclosure include Fc polypeptides and retain Fc-mediated effector functions. In various cases, bispecific antigen binding proteins are bispecific monoclonal antibodies formed by, for example, chemical crosslinking of two monoclonal antibodies (mabs) or by knob and hold technology. In exemplary aspects, bispecific antigen binding proteins are made by "knobs-into-holes" technology, in which different mutations are introduced into two CH3 domains to force the H chain to heterodimerize, thereby producing asymmetric antibodies. "Knob" mutations are generated in one HC, and "hole" mutations are generated in another HC to promote heterodimerization. In exemplary aspects, bispecific antigen binding proteins are bispecific antibodies produced by four-source hybridoma technology, which is based on somatic cell fusion of two different hybridoma cells to produce monoclonal antibodies with desired specificity. Zhang et al., 2017, supra. In exemplary aspects, the bispecific antigen binding protein is a crossMab, ortho-Fab IgG, DVD-Ig, two-in-one IgG, IgG-scFv and scFv2-Fc (Kontermann and Brinkmann, 2015, supra). In various aspects, the bispecific antigen binding protein is an Ig-scFv fusion, in which a new antigen binding portion is added to a full-length IgG, resulting in a fusion protein with tetravalence for two different antigens, such as an IgG C-terminal scFv fusion and an IgG N-terminal scFv fusion. In an exemplary case, the bispecific antigen binding protein is a dual variable domain IgG (DVD-IgG), in which the LC and HC variable regions of an IgG specific for one antigen are fused to the N-terminal LC and HC variable regions of an IgG specific for a second antigen via a linker to form a DVD-IgG. In exemplary aspects, the bispecific antigen binding protein is a diabody-Fc fusion, which involves replacing the Fab fragment of an IgG with a bispecific diabody

[0087] In alternative cases, the bispecific antigen-binding proteins of the present disclosure do not include Fc polypeptides. In exemplary aspects, the bispecific antigen-binding proteins include the variable domains of each parent monoclonal antibody, and the linker is cloned and connected to form a single-chain bispecific antibody. In exemplary aspects, the bispecific antigen-binding proteins are tandem scFv, double antibody formats, single-chain double antibodies, tandem double antibodies (TandAb), dual-affinity retargeting molecules (dual-affinity retargeting molecule, DART), dock-and-lock (DNL) and nano antibodies (Fan et al., J Hematol Oncol. 2015; 8: 130). In various aspects, the bispecific antigen-binding proteins are bispecific F (mab 1 )2, scFv, bispecific diabody (BsDb), single-chain bispecific diabody (scBsDb), single-chain bispecific tandem variable domain (scBsTaFv), dock-and-lock trivalent Fab (DNL-(Fab)3), single domain antibody (sdAb) or bispecific single domain antibody (BssdAb). In exemplary aspects, the bispecific antigen binding protein is a tandem scFv comprising two scFv fragments connected by an additional peptide linker such as a glycine-serine repeat motif. Optionally, the tandem scFv comprises the following structure: VL A - Connector 1 – VH A - Connector 2 – VH B - Connector 3 – VL B (VL and VH are derived from single-chain antibody fragments; A and B represent parent monoclonal antibodies A and B). In an exemplary aspect, the bispecific antigen-binding protein is a TandAb, which comprises two pairs of VL and VH domains connected into a single polypeptide chain (Reusch et al., MAbs. 2015; 7(3): 584-604). The two polypeptide products dimerize in a head-to-tail manner and form a homodimer with a larger molecular weight (about 105 kDa) after expression. In an exemplary aspect, the bispecific antigen-binding protein is a protein produced using crossMab technology, which is described in PNAS 108(27): 11187-92(2011). CrossMab does not have any chemical linkers or connectors and is produced by a method of forcing the correct light chains to associate in a bispecific heterodimeric IgG antibody. In exemplary aspects, the CrossMab is a bivalent (1+1), trivalent (2+1) and tetravalent (2+2) bispecific crossMab, or a non-Fc tandem antigen binding fragment (Fab) based crossMab. In exemplary cases, the crossMab is a crossMab Fab 、crossMab VH-VLor crossMab CH1-CL .

[0088] In exemplary aspects, the bispecific antigen-binding protein comprises a single domain antibody or nanobody comprising a single monomeric variable antibody domain. Optionally, the variable domain is based on a heavy chain variable domain. In alternative aspects, the variable domain is based on a light chain variable domain.

[0089] In exemplary aspects, the bispecific antigen binding protein is a bispecific T cell engager or BiTE is a bivalent small molecule that contains only the variable region of an antibody in the form of scFv connected by a flexible peptide linker. In exemplary aspects, the bispecific antigen-binding protein includes scFV, and the scFV includes the LC and HC variable regions of the currently disclosed CDH17 antibody and the LC and HC variable regions of the second antibody specific for the second antigen. In some embodiments, BiTE includes the LC and HC variable regions of the second antibody specific for CD3. In some embodiments, CD3 is CD3E. In some embodiments, BiTE is 07-0653-h43Bs, 07-0646-h7Bs or 07-0663-h7Bs. In some embodiments, 07-0653-h43Bs, 07-0646-h7Bs and 07-0663-h7Bs respectively include the amino acids provided in SEQ ID NO: 100, 99 and 101. In some embodiments, 07-0653-h43Bs, 07-0646-h7Bs, and 07-0663-h7Bs can be encoded by exemplary nucleic acid molecules comprising sequences provided as SEQ ID NOs: 106, 105, and 107, respectively. In some embodiments, the BiTE can also comprise a detectable marker, which can facilitate detection and / or purification of the BiTE. In some such embodiments, the BiTE also comprises a C-terminal (HIS)6 epitope tag. In exemplary cases, the bispecific antigen binding protein is a dual affinity redirected (DART) with Different from DART, the covalent connection between the two chains limits the freedom of the antigen binding site. Therefore, DART is compact and can form stable contacts between target cells and effector cells. DART contains two engineered Fv fragments, and the VH of these Fv fragments themselves is exchanged with the VH of another fragment. The mutually exchanged Fv domains advantageously release the variant fragments from the conformational constraints of the short connecting peptide.

[0090] In an exemplary aspect, the bispecific antigen binding protein is a HSABody, which comprises two scFvs fused to a modified HSA. HSABody is described in McDonagh et al., Mol Cancer Ther. 2012; 11(3):582-93.

[0091] Thus, in exemplary aspects, the bispecific antigen binding protein comprises an antigen binding fragment of any of the currently disclosed CDH17 antibodies. In exemplary aspects, the antigen binding fragment is a Fab. In exemplary aspects, the bispecific antigen binding protein comprises a F(ab) of any of the currently disclosed CDH17 antibodies. 2 '. In exemplary aspects, the bispecific antigen binding protein comprises an scFv comprising the LC and HC variable regions of any of the currently disclosed CDH17 antibodies. In various aspects, the antigen binding fragment is based on the heavy chain variable region, and in other aspects, the antigen binding fragment is based on the light chain variable region. In exemplary aspects, the antigen binding fragment comprises at least a portion of both the HC variable region and the LC variable region. In exemplary aspects, the bispecific antigen binding protein comprises at least one of the LC or HC variable regions of the currently disclosed CDH17 antibody (if not both) and at least one of the LC and HC variable regions of a second antibody having specificity for a second antigen (if not both). In exemplary cases, the bispecific antigen binding protein comprises an scFV comprising the LC and HC variable regions of the currently disclosed CDH17 antibody and the LC and HC variable regions of a second antibody having specificity for a second antigen.

[0092] Nucleic Acids

[0093] The present disclosure also provides nucleic acids comprising nucleotide sequences encoding antigen-binding proteins of the present disclosure. As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to polymers of DNA or RNA, or modified forms thereof, which may be single-stranded or double-stranded, synthesized or obtained from natural sources (e.g., isolated and / or purified); it may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, rather than phosphodiesters found between nucleotides of unmodified oligonucleotides. Nucleic acids may include any nucleotide sequence encoding any of the antigen-binding proteins of the present disclosure.

[0094] The present invention also provides nucleic acid molecules encoding the amino acid sequence corresponding to the antigen-binding protein of the present invention. In some embodiments, the nucleic acid molecule is DNA (e.g., cDNA) or a hybrid thereof. Alternatively, the molecule is RNA or a hybrid thereof.

[0095] In some aspects, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) molecules produced by replication of the molecules described in (i) above. For purposes herein, replication can be in vitro replication or in vivo replication.

[0096] Any nucleic acid disclosed herein can be subjected to codon optimization. Codon usage within a gene helps determine the achievable protein expression level. Certain sequences can be more easily translated by certain hosts, so selecting appropriate codons for a given host may be necessary for maximizing expression. Methods for optimizing codons are well known in the art. For example, there are many online tools, including the World Wide Web codonstatsdb.unr.edu (see Subramanian et al. (2022) Mol Biol Evol 3; 39(8): msac157).

[0097] In some aspects, nucleic acids are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al., supra; and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or nucleotides that have undergone various modifications designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed after hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl braid, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, braided glycoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0098] Carrier

[0099] In some aspects, the nucleic acid of the present disclosure is incorporated into a vector. In this regard, the present disclosure provides a vector comprising any one of the currently disclosed nucleic acids. In various aspects, the vector is a recombinant expression vector. For the purposes of this article, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct, when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is contacted with a cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell, it allows the host cell to express mRNA, protein, polypeptide or peptide. The vector of the present disclosure is not naturally occurring as a whole. However, each part of the vector may be naturally occurring. The currently disclosed vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and may contain natural, non-natural or altered nucleotides. The vector may contain naturally occurring or non-natural internucleotide linkages, or two types of linkages. In some aspects, the altered nucleotides or non-natural internucleotide linkages do not hinder the transcription or replication of the vector.

[0100] The carrier of the present disclosure can be any suitable carrier, and can be used for transduction, transformation or transfection of any suitable host. Suitable carriers include carriers designed for reproduction and amplification or for expression or both, such as plasmids and viruses. The carrier can be an expression vector based on a plasmid. In various aspects, the carrier is selected from the group consisting of: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJoIIa, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA). Phage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149, can also be used. Examples of plant expression vectors include pBIO1, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some aspects, the vector is a viral vector, such as a retroviral vector. In various aspects, the vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia virus vector or a lentiviral vector. See, for example, Howarth et al., Cell Biol. Toxicol. 26 (1): 1-20 (2010). In various aspects, the vector is a baculovirus vector that infects arthropods such as insects. In various aspects, the baculovirus vector is Autographacalifornica multiple nuclear virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV). See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013); Miller, Bioessays 11(4):91-96 (1989); Atkinson et al., Pestic Sci 28:215-224 (1990).

[0101] The vectors disclosed herein can be prepared using standard recombinant DNA techniques such as those described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, CoIE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0102] In some aspects, the vector contains regulatory sequences, such as transcriptional and translational start and stop codons, which are specific for the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA or RNA based.

[0103] The vector may include one or more marker genes to select transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc.; supplementation in auxotrophic hosts to provide original nutrition, etc. Suitable marker genes for currently disclosed expression vectors include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0104] The carrier can comprise a natural or standard promoter, which is operably connected to a nucleotide sequence of a coded polypeptide (including its functional part and functional variant), or is connected to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the polypeptide. The selection of promoters, such as strong promoters, weak promoters, inducible promoters, tissue-specific promoters and development-specific promoters, are all within the ordinary skill of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the skill of the technician. Promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, a RSV promoter and a promoter found in the long terminal repeat sequence of a mouse stem cell virus.

[0105] Host cells

[0106] Provided herein are host cells comprising nucleic acids or vectors disclosed herein. As used herein, the term "host cell" refers to any type of cell that may contain a currently disclosed vector and may produce an expression product (e.g., mRNA, protein) encoded by a nucleic acid. In some aspects, the host cell is an adherent cell or a suspension cell, i.e., a cell that grows in suspension. In various aspects, the host cell is a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as a human body. The host cell may be any cell type, may be derived from any type of tissue, and may be at any stage of development.

[0107] In various aspects, the antigen binding protein is a glycosylated protein and the host cell is a cell capable of glycosylation. In various aspects, the cell capable of glycosylation is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel et al., Front Immunol 4:217 (2013). In various aspects, the eukaryotic cell is a mammalian cell. In various aspects, the mammalian cell is a non-human mammalian cell. In some aspects, the cell is a Chinese hamster ovary (CHO) cell and its derivatives (e.g., CHO-K1, CHO pro-3), a mouse myeloma cell (e.g., NS0, GS-NS0, Sp2 / 0), a cell engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), a human embryonic kidney 293 (HEK293) cell or its derivatives (e.g., HEK293T, HEK293-EBNA), an African green monkey kidney cell (e.g., COS cell, VERO cell), a human cervical cancer cell (e.g., HeLa), a human skeletal osteosarcoma epithelial cell U2-OS, adenocarcinoma human alveolar basal epithelial cell A549, a human fibrosarcoma cell HT1080, a mouse brain tumor cell CAD, an embryonic carcinoma cell P19, a mouse embryonic fibroblast NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse B myeloma J558L or baby hamster kidney (BHK) cells (Gaillet et al., 2007; Khan, Adv Pharm Bull 3(2):257-263 (2013)).

[0108] For amplification or replication of the vector, in some aspects, the host cell is a prokaryotic cell, such as a bacterial cell.

[0109] The disclosure also provides a cell group, the cell group comprises at least one host cell described herein. In some aspects, the cell group is a heterogeneous colony, which includes a host cell containing the vector, and at least one other cell that does not include any vector. Or, in some aspects, the cell group is a substantially homogeneous colony, wherein the colony mainly includes a host cell containing a vector (e.g., substantially composed of a host cell containing a vector). In some aspects, the colony is a clonal colony of cells, wherein all cells of the colony are clones of a single host cell comprising a vector, so that all cells of the colony include the vector. In various embodiments of the disclosure, the cell group is a clonal colony comprising a host cell, and the host cell includes a vector described herein.

[0110] Manufacturing method

[0111] Also provided herein is a method for producing an antigen binding protein that binds to CDH17. In various embodiments, the method includes culturing a host cell comprising a nucleic acid in a cell culture medium, the nucleic acid comprising a nucleotide sequence encoding an antigen binding protein described herein; and harvesting the antigen binding protein from the cell culture medium. The host cell may be any host cell described herein. In various aspects, the host cell is selected from the group consisting of: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cell includes culturing the host cell in a growth medium to support the growth and expansion of the host cell. In various aspects, the growth medium increases cell density, culture viability, and productivity in a timely manner. In various aspects, the growth medium contains amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and attachment factors. In various aspects, the growth medium is a chemically completely defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to providing nutrients, the growth medium also helps maintain pH and osmotic pressure. Several growth media are commercially available and described in the art. See, e.g., Arora, “Cell Culture Media: A Review” MATERMETHODS 3: 175 (2013).

[0112] In various aspects, the method includes culturing the host cell in a feed medium. In various aspects, the method includes culturing in a feed medium in a fed batch mode. The production methods of recombinant proteins are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production" MAbs2(5):466–477 (2010).

[0113] The method for preparing an antigen binding protein may include one or more steps of purifying the protein from a cell culture or its supernatant and preferably recovering the purified protein. In various aspects, the method includes one or more chromatography steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In various aspects, the method includes purifying the protein using a protein A affinity chromatography resin.

[0114] In various embodiments, the method further comprises the step of formulating the purified protein, thereby obtaining a formulation comprising the purified protein. These steps are described in the following literature: Formulation and Process Development Strategies for Manufacturing, Jameel and Hershenson, eds., John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0115] In various aspects, the antigen binding protein connected to the polypeptide and the antigen binding protein is part of a fusion protein. Therefore, the present disclosure also provides a method for producing a fusion protein, the fusion protein comprising an antigen binding protein bound to CDH17. In various embodiments, the method includes culturing a host cell comprising a nucleic acid in a cell culture medium, the nucleic acid comprising a nucleotide sequence encoding a fusion protein described herein; and harvesting the fusion protein from the cell culture medium.

[0116] Conjugate

[0117] The present disclosure also provides an antigen binding protein attached, connected or conjugated to a second portion (e.g., a heterologous portion, a conjugated portion). Therefore, the present disclosure provides a conjugate comprising an antigen binding protein and a heterologous portion. As used herein, the term "heterologous portion" is synonymous with "conjugate portion" and refers to any molecule (chemical or biochemical, naturally occurring or non-encoded) that is different from the antigen binding protein of the present disclosure. Various heterologous portions include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines) or diagnostic agents.

[0118] In some embodiments, the heterologous moiety is a detectable label. Examples of detectable labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and / or radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin (PE); examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or3 H. As used herein, the term "labeling" with respect to an antibody is intended to encompass direct labeling of an antibody by coupling (i.e., physically linking) a detectable substance such as a radioactive agent or fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine green (Cy5)) to the antibody, as well as indirect labeling of an antibody by reactivity with a detectable substance. For example, an antibody can be labeled with a nucleic acid sequence that can be amplified and detected, or with an antisense oligonucleotide that reduces the expression of a specific gene, so that expression can be subsequently detected and measured.

[0119] In some embodiments, the heterologous moiety is a polymer. The polymer can be branched or unbranched. The polymer can have any molecular weight. In some embodiments, the average molecular weight of the polymer is between about 2kDa and about 100kDa (the term "about" means that in the preparation of a water-soluble polymer, the weight of some molecules will be greater than the molecular weight, and the weight of some molecules will be less than the molecular weight). In some aspects, the average molecular weight of the polymer is between about 5kDa and about 50kDa, between about 12kDa and about 40kDa, or between about 20kDa and about 35kDa.

[0120] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. In some embodiments, the polymer is water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, when, for example, the composition is used for therapeutic purposes, the polymer is pharmaceutically acceptable. In addition, in some aspects, the polymer is a mixture of polymers, such as a copolymer, a block copolymer.

[0121] In some embodiments, the polymer is selected from the group consisting of: polyamides; polycarbonates; polyalkylenes and their derivatives, including polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates; polymers of acrylic and methacrylates, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyvinyl polymers, including poly( Vinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly(vinyl acetate) and polyvinyl pyrrolidone; polyglycolide; polysiloxanes; polyurethane and copolymers thereof; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate and cellulose sulfate sodium salt; polypropylene; polyethylene, including poly(ethylene glycol), poly(ethylene oxide) and poly(ethylene terephthalate); and polystyrene.

[0122] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass any form of PEG that can be used to derivatize other proteins, such as mono(C1-C10) alkoxy polyethylene glycol or aryloxy polyethylene glycol. PEG is a linear or branched neutral polyether with a variety of molecular weights and is soluble in water and most organic solvents.

[0123] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.

[0124] In some embodiments, the heterologous moiety is a lipid. In some embodiments, the lipid is a fatty acid, an eicosanoid, a prostaglandin, a leukotriene, a thromboxane, N-acylethanolamine), a glycerolipid (e.g., monosubstituted, disubstituted, trisubstituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., a steroid, cholesterol), a prenol lipid, a glycolipid or a polyketide, an oil, a wax, cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, a phospholipid.

[0125] In some embodiments, the heterologous portion is a therapeutic agent. The therapeutic agent can be any therapeutic agent known in the art. Examples of therapeutic agents encompassed herein include, but are not limited to, natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomimetics, angiotensin converting enzyme (ACE) inhibitors, diuretics, myocardial contractility enhancers (inotrope), cholesterol and triglyceride lowering agents, bile acid sequestrants, fibrates, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, nicotinic acid derivatives, antiadrenergic drugs, α-adrenergic blockers, centrally acting antiadrenergics, vasodilators, potassium sparing agents, thiazides and related drugs, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, α-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestins, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation induction agents, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, bulking agents forming agent), laxative, antiperistaltics, flora modifier, enterosorbent, intestinal anti-infective agent, anorexic, anti-cachexia agent, antibulimic, appetite suppressant, slimming agent, antacid, upper gastrointestinal tract agent, anticholinergic agent, aminosalicylic acid derivative, biological response modifier, corticosteroid, antispasmodic, 5-HT4 partial agonist, antihistamine, cannabinoid, dopamine antagonist, serotonin antagonist, cytoprotective agent, histamine H2 receptor antagonist, mucosal protectant, proton pump inhibitor, Helicobacter pylori (H.pylori eradication therapy, erythropoiesis stimulating agents, hematopoietic agents, anemia agents, heparin, antifibrinolytic agents, hemostatic agents, coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane-A2 inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, drugs involved in preventive therapy, AIDS-related infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma sarcoma), aminoglycosides, carbapenems, cephalosporins, glycopoptides, lincosamides, macrolies, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and its derivatives, tetracyclines, anthelmintics, amebicies, biguanides, cinchona alkaloids alkaloids), folic acid antagonists, quinoline derivatives, Pneumocystis carinii therapy, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy, barbiturates and their derivatives, benzodiazepines. benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigraine preparations, biological response modifiers, carbamic acid eaters, tricyclic derivatives, depolarizing agents, nondepolarizing agents, neuromuscular paralytic agents, CNS stimulants, dopaminergic reagents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, ethyleneimines, imidazotetrazines, nitrogen mustard analogs mustardanalog), nitrosoureas, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycinds, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and their analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors, antineoplastic agents, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepines Derivatives, dibenzothiazepine (dibenzothiazepine) derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepines derivatives, thioxanthene derivatives, allergen extracts, nonsteroidal drugs, leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, aminobenzoates, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride, hydrochloride), antipyretics, hynotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, vitamin K analogs and antagonists.

[0126] The antigen binding proteins disclosed herein can be conjugated with one or more cytokines and growth factors that effectively inhibit tumor metastasis, and wherein the cytokines or growth factors have been shown to have an anti-proliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein-16, bone morphogenetic protein-17, bone morphogenetic protein-18, bone morphogenetic protein-19, bone morphogenetic protein-20, bone morphogenetic protein-21, bone morphogenetic protein-22, bone morphogenetic protein-23, bone morphogenetic protein-24, bone morphogenetic protein-25, bone morphogenetic protein-26, bone morphogenetic protein-27, bone morphogenetic protein-28, bone morphogenetic protein-29, bone morphogenetic protein-30, bone morphogenetic protein-31, bone morphogenetic protein-32, bone morphogenetic protein-33, bone morphogenetic protein-34, bone morphogenetic protein-35, bone morphogenetic protein-36, bone morphogenetic protein-37 leukocyte receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, β-endothelial cell growth factor, endothelin 1, epithelial cell-derived neutrophil attractant, glial cell line-derived neutrophil receptor α1, glial cell line-derived neutrophil receptor α2, Growth-related protein, growth-related protein alpha, growth-related protein beta, growth-related protein gamma, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor receptor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, chimeric proteins and biologically or immunologically active fragments thereof.

[0127] In some embodiments, the conjugate comprises antigen-binding proteins described herein and cytotoxic agents.Cytotoxic agents are any molecules (chemical or biochemical) that are toxic to cells.In some respects, when cytotoxic agents are conjugated with antigen-binding proteins of the present disclosure, the result obtained is synergistic.That is to say, the effectiveness of the combination therapy of antigen-binding proteins and cytotoxic agents is synergistic, that is, its effectiveness is greater than the effectiveness expected by the addition of its respective individual effects.Therefore, the dosage of cytotoxic agents can be reduced, and therefore the risk of toxicity problems and other side effects is reduced simultaneously.In some embodiments, cytotoxic agents are chemotherapeutic agents.Chemotherapeutic agents are known in the art, including but not limited to platinum coordination compounds, topoisomerase inhibitors, antibiotics, anti-mitotic alkaloids and difluoronucleosides, as described in U.S. Patent No. 6,630,124.

[0128] In some embodiments, the chemotherapeutic agent is a platinum coordination compound.The term "platinum coordination compound" refers to any platinum coordination compound that provides platinum in ionic form and inhibits tumor cell growth.

[0129] In some embodiments, the platinum coordination compound is cis-diamine dihydrate platinum (II)-ion; chloro(diethylenetriamine)-platinum (II) chloride; dichloro(ethylenediamine)-platinum (II); diammine(1,1-cyclobutanedicarboxyl)platinum (II) (carboplatin); spiroplatin; isopropylplatin; diammine(2-ethylmalonic acid)-platinum (II); ethylenediaminemalonate platinum (II); hydrated (1,2-diaminodicyclohexane)-sulfate platinum (II); (1,2-diaminocyclohexane)malonate platinum (II); (4-carboxyphthalate) (1,2-diaminocyclohexane) platinum (II); (1,2-diaminocyclohexane)-(isocitrate) platinum (II); (1,2-diaminocyclohexane) cis(pyruvate) platinum (II); (1,2-diaminocyclohexane) oxalate platinum (II); ormaplatin; and tetraplatinum.

[0130] In some embodiments, cisplatin is a platinum coordination compound used in the compositions and methods of the invention. Cisplatin is available from Bristol Myers-Squibb Corporation under the name PLATINOL TMCisplatin, or cis-dichlorodiamine platinum II, has been successfully used as a chemotherapeutic agent to treat various human solid malignancies. Recently, other diaminoplatinum complexes have also been shown to be effective as chemotherapeutic agents for the treatment of various human solid malignancies. Such diaminoplatinum complexes include, but are not limited to, spiroplatin and carboplatin. Although cisplatin and other diaminoplatinum complexes have been widely used as human chemotherapeutic agents, they must be delivered at high dose levels, which may lead to toxicity problems such as kidney damage.

[0131] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can change the topological structure of DNA in eukaryotic cells. They are essential for cell function and cell proliferation. In general, there are two types of topoisomerases in eukaryotic cells: type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of about 100,000. This enzyme binds to DNA and introduces transient single-strand breaks, unwinds the double helix (or unwinds it), and then reseals the break before dissociating from the DNA chain. Recently, various topoisomerase inhibitors have shown clinical efficacy in the treatment of human patients with ovarian cancer, esophageal cancer or non-small cell lung cancer.

[0132] In some aspects, the topoisomerase inhibitor is camptothecin or a camptothecin analog. Camptothecin is a water-insoluble cytotoxic alkaloid produced by Camptotheca accuminata, which originates in China, and Nothapodytes foetida, which originates in India. Camptothecin exhibits tumor cell growth inhibitory activity against a variety of tumor cells. Camptothecin analog compounds are typically specific DNA topoisomerase I inhibitors. The term "topoisomerase inhibitor" refers to any tumor cell growth inhibitory compound structurally related to camptothecin. Camptothecin analog compounds include, but are not limited to, topotecan, irinotecan, and 9-aminocamptothecin.

[0133] In other embodiments, the cytotoxic agent is a camptothecin analog claimed or described in the following documents to inhibit the growth of any tumor cell: U.S. Patent No. 5,004,758 issued on April 2, 1991 and European Patent Application No. 88311366.4 published on June 21, 1989 as 20' publication No. EP 0 321 122; U.S. Patent No. 4,604,463 issued on August 5, 1986 and European Patent Application Publication No. EP 0 137 145 published on April 17, 1985; U.S. Patent No. 4,473,692 issued on September 25, 1984 and European Patent Application Publication No. EP 0 074 256 published on March 16, 1983; U.S. Patent No. 4,545,880 issued on October 8, 1985 and European Patent Application Publication No. EP 0 074 256 published on March 16, 1983 256; European Patent Application Publication No. EP 0 088 642 published on September 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1, pp. 28-30, especially the compound known as CPT-11. CPT-11 is a camptothecin analog having a 4-(piperidino)-piperidine side chain attached at C-10 of 10-hydroxy-7-ethylcamptothecin via a carbamate linkage. CPT-11 is currently undergoing human clinical trials and is also known as irinotecan; Wani et al., J. Med. Chem., 23, 554 (1980); Wani et al., J. Med. Chem., 30, 1774 (1987); U.S. Patent No. 4,342,776, issued August 3, 1982; U.S. Patent Application Serial No. 581,916, filed September 13, 1990, and European Patent Application Publication No. EP 418 099, published March 20, 1991; U.S. Patent No. 4,513,138, issued April 23, 1985, and European Patent Application Publication No. EP 0 074 770, published March 23, 1983; U.S. Patent No. 4,399,276, issued August 16, 1983, and European Patent Application Publication No. 0 056, published July 28, 1982 692; the entire disclosure of each of which is hereby incorporated by reference. All of the camptothecin analog compounds listed above are commercially available and / or can be prepared using conventional techniques (including those described in the references listed above). The topoisomerase inhibitor can be selected from the group consisting of topotecan, irinotecan and 9-aminocamptothecin.

[0134] In some embodiments, the camptothecin analog is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by the hydrolysis of irinotecan by carboxylesterase. In some embodiments, SN-38 has one of the following structures:

[0135]

[0136] SN-38 is described in the following patents: U.S. Patent Application Serial No. 7,999,083; U.S. Patent Application Serial No. 8,080,250; U.S. Patent Application Serial No. 8,759,496; U.S. Patent Application Serial No. 8,999,344; U.S. Patent Application Serial No. 10,195,288; and U.S. Patent Application Serial No. 9,808,537.

[0137] In some embodiments, the camptothecin analog is exatecan methanesulfonate. Exatecan methanesulfonate is a water-soluble camptothecin (CPT) that exhibits more potent topoisomerase I inhibitory activity and anti-tumor activity than other CPT analogs. In addition, exatecan is effective against multidrug-resistant cells mediated by p-glycoprotein (P-gp).

[0138] In some embodiments, the camptothecin analog is deruxtecan (Dxd), which is a potent derivative of exotecan with a topoisomerase I inhibitory potency 10 times higher than SN-38. In some embodiments, Dxd has the following structure:

[0139]

[0140] Dxd has been described in the following patents: US Patent Application Serial No. 6,407,115; US Patent Application Serial No. 10,195,288; US Patent Application Serial No. 9,808,537; and US Patent Application Serial No. 6,407,115.

[0141] The preparation of various compounds of camptothecin analogs (including pharmaceutically acceptable salts, hydrates and solvates thereof) and the preparation of oral and parenteral pharmaceutical compositions containing such camptothecin analogs and pharmaceutically acceptable inert carriers or diluents are described in detail in U.S. Patent No. 5,004,758, issued on April 2, 1991, and European Patent Application No. 88311366.4, published on June 21, 1989 as Publication No. EP 0 321122, the teachings of which are incorporated herein by reference.

[0142] In other embodiments of the invention, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0143] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. In general, antimitotic alkaloids can be extracted from Cantharanthus roseus, and it has been shown that they can be used as effective anticancer chemotherapeutic agents. Many semi-synthetic derivatives have been studied in chemistry and pharmacology (see O. Van Tellingen et al., Anticancer Research, 12, 1699-1716 (1992)). The antimitotic alkaloids of the present invention include but are not limited to vinblastine, vincristine, vindesine, taxol and vinorelbine. The latter two antimitotic alkaloids can be purchased from Eli Lilly and Company and Pierre Fabre Laboratories respectively (see U.S. Patent No. 5,620,985). In some embodiments, the antimitotic alkaloid is vinorelbine.

[0144] In other embodiments of the present invention, the chemotherapeutic agent is a difluoro nucleoside. It is known in the art that 2'-deoxy-2', 2'-difluoro nucleosides have antiviral activity. These compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808614. European Patent Application Publication 184,365 discloses that these same difluoro nucleosides have oncolytic activity. In certain aspects, the 2'-deoxy-2', 2'-difluoro nucleosides used in the compositions and methods of the present invention are 2'-deoxy-2', 2'-difluoro cytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine is commercially available, or can be synthesized in a multistep process according to the disclosure and teachings in U.S. Patent Nos. 4,526,988, 4,808,614 and 5,223,608, the teachings of which are incorporated herein by reference.

[0145] In various aspects, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization, destabilizing microtubules, or changing microtubule dynamics, such as maytansinoid or its derivatives (e.g., DM1 or DM4), auristatin or its derivatives. In various cases, the chemotherapeutic agent is auristatin. For example, in some aspects, auristatin is dolastatin, monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE) or PF-06380101. Auristatin has been described in the art. See, for example, Maderna, A.; et al., Mol Pharmaceutics 12 (6): 1798-1812 (2015). In various aspects, the conjugate comprises an antibody of the present disclosure combined with MMAE. Optionally, the conjugate comprises a linker. In some aspects, the linker comprises a cleavable linking portion. In various cases, the conjugate comprises an antibody of the present disclosure linked to a linker, which is linked to a cathepsin-cleavable linker, which is in turn linked to a spacer, which is linked to MMAE. In some aspects, the linker is linked to the antibody via a Cys residue in the Fc region of the antibody. In exemplary aspects, the linker comprises a structure of Formula I:

[0146]

[0147] In exemplary aspects, the cathepsin-cleavable linker comprises the structure of Formula II:

[0148]

[0149] In exemplary aspects, the spacer comprises the structure of Formula III:

[0150]

[0151] In some embodiments, MMAE has the following structure:

[0152]

[0153] The present disclosure also provides the following conjugates, the conjugates comprising the antigen-binding proteins of the present disclosure connected to the polypeptide, thereby making the conjugates a fusion protein. Therefore, the present disclosure provides fusion proteins, the fusion proteins comprising the antigen-binding proteins of the present disclosure connected to the polypeptide. In various embodiments, the polypeptide is a diagnostic marker, such as a fluorescent protein, such as green fluorescent protein, or other labels, such as Myc tags. In various aspects, the polypeptide is one of the cytokines, lymphokines, growth factors or other hematopoietic factors listed above.

[0154] Connectors

[0155] In some embodiments, the conjugate is directly connected to the heterologous part. In alternative embodiments, the conjugate includes a joint, and the joint joins the compound of the present disclosure to the heterologous part. In some aspects, the joint includes 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms or 10 to 20 atoms long atomic chain. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the joint main chain are selected from the group consisting of C, O, N and S. Chain atoms and joints can be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the joint provides a functional group that is easily cracked by an enzyme or other catalyst or hydrolysis condition found in a target tissue or organ or cell. In some embodiments, the length of the joint is long enough to reduce the possibility of steric hindrance. In some embodiments, the joint is an amino acid or peptidyl joint. Such peptidyl joints can be of any length. Various linkers are about 1 to 50 amino acids long, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids long.

[0156] A variety of suitable linkers are known in the art. The linker can be cleavable (cleavable linker), for example, under physiological conditions, such as cleavable under intracellular conditions, so that the linker is cleaved and releases the drug in the intracellular environment. Alternatively, the linker can be cleaved under extracellular conditions, such as cleavage outside the tumor cell or near the mass, so that the linker is cleaved and releases the drug that preferentially penetrates into the tumor cell. In other embodiments, the linker is non-cleavable (non-cleavable linker), and the drug is released, for example, by antibody degradation.

[0157] The linker can be bonded to a chemically reactive group on the antibody portion, such as a free amino group, an imino group, a hydroxyl group, a thiol group, or a carboxyl group (e.g., to the N-terminus or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, to the sulfhydryl group of one or more cysteinyl residues, or to the hydroxyl group of one or more serine or threonine residues). The site to which the linker is bound can be a natural residue in the amino acid sequence of the antibody portion, or it can be introduced into the antibody portion, such as by recombinant DNA technology (e.g., by introducing a cysteine ​​or protease cleavage site in the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The site to which the linker is bound can also be a non-natural amino acid. The site to which the linker is bound can also be a polysaccharide on the antibody.

[0158] Typically, the linker is substantially inert under the conditions in which the two groups it connects are located. The term "bifunctional cross-linker," "bifunctional linker," or "cross-linking agent" refers to a modifier having two reactive groups at each end of the linker, whereby one reactive group can first react with a cytotoxic compound to provide a compound with a linker moiety and a second reactive group, which can then react with the antibody. Alternatively, one end of the bifunctional cross-linker can first react with the antibody to provide an antibody with a linking moiety and a second reactive group, which can then react with the cytotoxic compound. The linking moiety can contain a chemical bond that allows the cytotoxic moiety to be released at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, protease / peptidase-labile bonds, and esterase-labile bonds. See, for example, U.S. Patent Nos. 5,208,020, 5,475,092, 6,441,163, 6,716,821, 6,913,748, 7,276,497, 7,276,499, 7,368,565, 7,388,026, and 7,414,073, each of which is incorporated herein by reference. In some embodiments, the bond is a disulfide bond, a thioether bond, and / or a protease / peptidase instability bond. Other joints that can be used in the present invention include non-cleavable joints (such as joints described in detail in US20050169933), charged joints, or hydrophilic joints (such as joints described in US2009 / 0274713, US2010 / 0129314, and WO 2009 / 134976), each of which is clearly incorporated herein by reference.

[0159] In some embodiments, the linker is a hydrophilic linker that imparts hydrophilicity to the conjugate. In some embodiments, the hydrophilic linker comprises polyethylene glycol (PEG). In some embodiments, the hydrophilic linker is CLA2. In some embodiments, the CLA2 linker has the following structure:

[0160]

[0161] CLA2 is described in US Pat. Nos. 8,080,250, 8,759,496, and 10,195,288, each of which is incorporated herein by reference.

[0162] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure:

[0163]

[0164] CL2E is described in US Pat. Nos. 8,080,250, 8,759,496, and 10,195,288, each of which is incorporated herein by reference.

[0165] In some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., lysosomes or endosomes or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular or extracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). In some embodiments, the peptide linker comprises at least two, at least three, at least four, or at least five amino acids long.

[0166] In some embodiments, the peptide linker is VC-PAB, which comprises valine and citrulline residues. In some such embodiments, the peptide linker is MC-VC-PAB. In some embodiments, the MC-VC-PAB linker has the following structure:

[0167]

[0168] MC-VC-PABs are described in U.S. Patent Nos. 7,659,241, 7,829,531, 6,884,869, 6,214,345, and 6,214,345, each of which is incorporated herein by reference.

[0169] In some embodiments, the peptide linker is glycine-glycine-phenylalanine-glycine (GGFG). In some such embodiments, the peptide linker is maleimidocaproylglycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure:

[0170]

[0171] MC-GGFG is described in US Pat. Nos. 9,808,537 and 10,195,288, each of which is incorporated herein by reference.

[0172] In other embodiments, the cleavable linker is pH-sensitive, that is, sensitive to hydrolysis at certain pH values. In some embodiments, the pH-sensitive linker can be hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that can be hydrolyzed in lysosomes can be used (see, e.g., U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661, each of which is incorporated herein by reference). These linkers are relatively stable under neutral pH conditions, such as under pH conditions in blood, but are unstable under pH values ​​lower than 5.5 or 5.0 (the approximate pH value of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether that is attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929, which is incorporated herein by reference).

[0173] In other embodiments, the linker can be cleaved under reducing conditions (e.g., a disulfide linker). Bifunctional cross-linkers capable of linking antibodies to cytotoxic compounds via disulfide bonds include, but are not limited to, N-succinimidyl-4-(4-nitropyridyl-2-dithio)butyrate, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB). Sulfo-SPDB is described, for example, in U.S. Pat. No. 8,236,319, which is incorporated herein by reference. Alternatively, cross-linking agents that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetyl succinic anhydride, may also be used. In other embodiments, the linker may contain a combination of one or more of the previously described peptide linkers, pH-sensitive linkers, or disulfide linkers.

[0174] A "heterobifunctional crosslinker" is a bifunctional crosslinker having two differently reactive groups. Heterobifunctional crosslinkers containing an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds to antibodies. Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazino terephthalate hydrochloride (SHTH), and succinimidyl hydrazinonicotinate hydrochloride (SHNH). Benzodiazepines with hydrazines of the present invention can also be used. Derivatives are used to prepare conjugates with acid-labile linkages. Examples of bifunctional cross-linking agents that can be used include succinimidyl-p-formyl benzoate (SFB) and succinimidyl-p-formyl phenoxy acetate (SFPA).

[0175] The linkers described herein can be used in any combination with the heterologous moieties described herein. In addition, the linkers described herein can have any chemically reactive moieties (e.g., maleimide, cysteine, etc.) that can react with any portion of the antigen-binding proteins of the present disclosure (e.g., amino acids, disulfide bonds, carbohydrates (e.g., carbohydrates from post-translational modifications), etc.). Typically, lysine or cysteine ​​on an antibody or antigen-binding protein (e.g., cysteine ​​obtained by reducing disulfide bonds (e.g., interchain or intrachain disulfide bonds from an antibody or antigen-binding protein) or an engineered unpaired cysteine) is used as a conjugation site. All of the above-listed linkers and heterologous moieties described herein are commercially available and / or can be prepared by conventional techniques (including the techniques described in the references listed above).

[0176] Conjugation

[0177] The ratio (HAR) of heterologous moiety to antigen binding protein represents the number of heterologous moieties connected on each antigen binding molecule. In some embodiments, the HAR range is 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2. In some embodiments, the HAR range is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, HAR is about 2, about 2.5, about 3, about 4, about 5 or about 6. In some embodiments, the HAR range is about 2 to about 4. HAR can be characterized by conventional methods, such as mass spectrometry, UV / Vis spectrum, ELISA determination and / or HPLC.

[0178] In some embodiments, the conjugate is a heteroconjugate (also referred to as "conventional"), in which the antigen binding protein is conjugated to different numbers of heterologous moieties. In some embodiments, the heteroconjugate follows a Gaussian distribution or quasi-Gaussian distribution of conjugates, wherein the distribution is centered around an average heterologous moiety loading value, wherein some antigen binding proteins have conjugated values ​​above the average and some antigen binding proteins have conjugated values ​​below the average.

[0179] In some embodiments, the conjugate is a homogeneous conjugate in which a majority of the antigen binding protein is conjugated to a defined number of heterologous moieties. In some embodiments, the homogeneous conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HARs. In some embodiments, the homogeneous conjugate comprises 2, 4, 6, or 8 HARs. In preferred embodiments, the homogeneous conjugate comprises 4 HARs. In other preferred embodiments, the homogeneous conjugate comprises 2 HARs. In some embodiments, the homogeneous conjugate comprises greater than or equal to 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the conjugate having a defined HAR. In some embodiments, the homogeneous conjugate comprises about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the conjugate with a defined HAR. In some embodiments, the homogeneous conjugate comprises at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the conjugate with a defined HAR. In some embodiments, the homogeneous conjugate comprises a HAR distribution that is non-Gaussian or quasi-Gaussian. In some embodiments, the homogeneity of the homogeneous conjugate is determined by a chromatogram, such as HPLC or any suitable chromatography. In some embodiments, the chromatogram is a HIC chromatogram. The homogeneous conjugate can be produced by site-specific conjugation.

[0180] In some embodiments, the heterologous moiety is conjugated to an antigen binding protein (e.g., an antibody) in a site-specific manner. Various site-specific conjugation methods are known in the art, such as thiomab or TDC or conjugation at unpaired cysteine ​​residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932; Dimasi et al. (2017) Mol. Pharm. 14:1501-1516; Shen et al. (2012) Nat. Biotechnol. 30:184-9); thiol bridges (Behrens et al. (2015) Mol. Pharm. 12:3986-98); conjugation at glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15; Dennler et al. (2014) Bioconjug Chem. 25:569-78); conjugation at engineered non-natural amino acid residues (Axup et al. (2012) Proc Natl Acad Sci USA 104-16101-6; Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71; Van Brunt et al. (2015) Bioconjug Chem 26:2249-60; Zimmerman et al. (2014) Bioconjug Chem 25:351-61); selenocysteine ​​conjugation (Li et al. (2017) Cell Chem Biol 24:433-442); glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5); conjugation at galactose or GalNAc analogs (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9; van Geel et al. (2015) Bioconjug Chem 26:2233-42); via glycan engineering (Zhou et al. (2014) Bioconjug Chem 25:510-20; Tang et al. (2017) Nat Protoc 12:1702-1721); via short peptide tags, such as engineered glutamine tags or sortase A-mediated transpeptidation (Strop et al. (2013) Chem Biol 20:161-7; Beerli et al. (2015) PLoS One 10:e0131177); and via aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5), each of which is incorporated herein by reference.

[0181] Unpredictable nature of conjugates (e.g. ADCs)

[0182] Based on the antibody profile or drug payload profile alone, it is impossible to predict in advance which antibody-drug conjugate will be safe and effective enough for clinical use. For example, a specific drug payload may work very well when conjugated to an antibody for one target, but when conjugated to an antibody for a different target, or even when combined with a different antibody for the same target, its effect may not be so good. The reasons why different antibody-drug conjugates exhibit different anti-tumor activities in vivo are not yet fully understood, making it impossible to make accurate predictions when designing new antibody-drug conjugates. It is speculated that the unpredictable interaction of multiple factors plays a role. These factors may include, for example, the binding affinity of the antibody-drug conjugate to the target antigen, the ability of the conjugate to penetrate solid tumors, and the circulation half-life of the tumor that is appropriately exposed without causing toxicity.

[0183] Antibody affinity alone is a good example of the complexity and unpredictability. Antibodies or antibody-drug conjugates with high affinity have better cellular uptake, resulting in higher levels of cytotoxic payload release inside the cell. Higher affinity is also known to enhance antibody-dependent cellular cytotoxicity (ADCC). All of these attributes favor the cell-killing properties of antibody-drug conjugates. However, it is well known that high affinity of antibodies or antibody-drug conjugates can prevent effective tumor penetration through the “antigenic barrier effect,” suggesting that in order to achieve strong anti-tumor activity in vivo, the affinity of the antibody-drug conjugate must be just right: not too high, not too low. To date, it is not known how to predict the most efficient or effective affinity level for an antibody-drug conjugate.

[0184] In addition, the mechanism of the linker and the payload alone cannot predict the in vivo anti-tumor activity. For example, O.Ab et al., Mol.Cancer Ther.14(&):1605-1613(2015), which is incorporated herein by reference, demonstrated that the same antibody conjugated to the same anti-tubulin toxin by different linkers exhibited distinct anti-tumor activity when tested in preclinical cancer models. This example is particularly surprising because the chemical structures of the two linkers are very similar. In addition, the linker present in the superior conjugate contains a hydrophilic part. Hydrophilic metabolites generally have lower membrane permeability and are thought to flow out of lysosomes (the site of conjugate degradation) more slowly, resulting in delayed anti-tubulin activity of the released payload. This finding advocates that payload delivery has "ideal" kinetics, but as of now, it is unclear what the contributing factors are to this kinetics. Adding to this complexity is an unresolved question: whether the ideal kinetics of payload delivery, even if determined for a specific cell type, is also applicable to all cell types. Therefore, it is impossible to predict the most effective in vivo anti-tumor activity only from the chemical composition of the linker or payload.

[0185] Compositions, pharmaceutical compositions and formulations

[0186] Provided herein are compositions comprising antigen-binding proteins, nucleic acids, carriers, host cells or conjugates as currently disclosed. In some respects, the compositions comprise antigen-binding proteins in a separated and / or purified form. In some respects, the compositions comprise antigen-binding proteins of the present disclosure of a single type (e.g., structure) or a combination comprising two or more antigen-binding proteins of the present disclosure, wherein the combination comprises two or more antigen-binding proteins of different types (e.g., structure).

[0187] In some aspects, the composition comprises an agent that enhances the chemical-physical characteristics of the antigen binding protein, such as by stabilizing the antigen binding protein at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation (e.g., oxidative protease-mediated degradation), increasing the half-life of the antigen binding protein, etc. In some aspects, the composition comprises any agent disclosed herein as a heterologous moiety or part of a conjugate, optionally admixed with an antigen binding protein of the disclosure or conjugated to an antigen binding protein.

[0188] In various aspects of the present disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the antigen binding proteins, nucleic acids, carriers, host cells or conjugates disclosed herein (hereinafter referred to as "active agents") are formulated into pharmaceutical compositions comprising active agents and pharmaceutically acceptable carriers, diluents or excipients. In this regard, the present disclosure also provides pharmaceutical compositions comprising active agents, which are intended to be administered to subjects, such as mammals.

[0189] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains an active agent at a concentration of about 0.001 to about 30.0 mg / ml (w / v).

[0190] In various aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solution; water; emulsions, such as oil / water emulsions or water / oil emulsions; and various types of wetting agents. The term also encompasses any agent approved by the U.S. federal government regulatory agency or listed in the U.S. Pharmacopoeia for use in animals (including humans).

[0191] The pharmaceutical composition may contain any pharmaceutically acceptable ingredient, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacers, alkalizers, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, bactericides, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubility enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, flavor enhancers, flavoring agents, glidants, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, tablet bases, pigments, plasticizers, polishing agents, preservatives, masking agents, skin penetrating agents, solubilizers, solvents, stabilizers, suppository bases, surface active agents, Agents, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxicants, viscosity enhancers, water absorbents, water miscible cosolvents, water softeners or wetting agents. See, for example, Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated herein by reference in its entirety.

[0192] In various aspects, the pharmaceutical composition comprises a formulation material that is non-toxic to the recipient at the dose and concentration employed. In a specific embodiment, the pharmaceutical composition comprises an active agent and one or more pharmaceutically acceptable salts; a polyol; a surfactant; an osmotic balance agent; a tonicity agent; an antioxidant; an antibiotic; an antifungal agent; a bulking agent; a lyoprotectant; a defoamer; a chelating agent; a preservative; a colorant; an analgesic; or an additional agent. In various aspects, optionally, in addition to one or more excipients, the pharmaceutical composition further comprises one or more polyols and / or one or more surfactants, including but not limited to pharmaceutically acceptable salts; an osmotic balance agent (tonicity agent); an antioxidant; an antibiotic; an antifungal agent; a bulking agent; a lyoprotectant; a defoamer; a chelating agent; a preservative; a colorant; and an analgesic.

[0193] In certain embodiments, pharmaceutical compositions may contain formulation materials used to alter, maintain or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride); the pharmaceutical composition may be a pharmaceutical composition comprising: ... See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (AR Genrmo, ed.), 1990, Mack Publishing Company, incorporated herein by reference.

[0194] The pharmaceutical composition can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition can be, for example, between about 4 or about 5 and about 8.0, or between about 4.5 and about 7.5, or between about 5.0 and about 7.5. In various embodiments, the pH of the pharmaceutical composition is between 5.5 and 7.5.

[0195] The present disclosure provides methods for manufacturing pharmaceutical compositions. In various aspects, the methods include combining an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharmaceutically acceptable carrier, diluent, or excipient.

[0196] Route of administration

[0197] For the purposes of the present disclosure, an active agent or a pharmaceutical composition comprising the active agent can be administered to a subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal or rectal administration. The following discussion of routes of administration is provided merely to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0198] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term "parenteral" means not passing through the digestive tract, but by some other route, such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agents of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or liquid mixture, including water; saline; aqueous dextrose and related sugar solutions; alcohols, such as ethanol or hexadecanol; glycols, such as propylene glycol or polyethylene glycol; dimethyl sulfoxide; glycerol; ketals, such as 2,2-dimethyl-153-dioxolane-4-methanol; ethers, poly(ethylene glycol) 400; oils; fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or detergent; a suspending agent, such as pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose; or an emulsifier, as well as other pharmaceutically acceptable adjuvants.

[0199] Oils that can be used in parenteral formulations include petrolatum, animal oils, vegetable oils or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Fatty acids suitable for parenteral formulations include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0200] Soaps suitable for use in parenteral formulations include fatty alkali metal salts, ammonium salts and triethanolamine salts, and suitable detergents include (a) cationic detergents such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0201] In some embodiments, the parenteral formulation contains a solution of the active agent of the present disclosure of about 0.5% to about 25% by weight. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is generally in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic matrix, the hydrophobic matrix being formed by the condensation of propylene oxide with propylene glycol. In some aspects, the parenteral formulation is present in a unit dose or multi-dose sealed container, such as an ampoule and a vial, and can be stored under freeze drying (lyophilization) conditions, requiring only the addition of a sterile liquid excipient, such as water for injection, before use. In some aspects, extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the kind previously described.

[0202] Injectable formulations are consistent with the present disclosure. The requirements for effective pharmaceutical carriers in injectable compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982); and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986), each of which is incorporated herein by reference).

[0203] dose

[0204] It is believed that the activating agents of the present disclosure can be used in methods for inhibiting tumor growth and other methods described herein, including methods for treating or preventing cancer. For the purposes of the present disclosure, the amount or dosage of the activating agent administered should be sufficient to produce, for example, a therapeutic or preventive response in a subject or animal within a reasonable time frame. For example, the dosage of the activating agent of the present disclosure should be sufficient to treat cancer described herein within a time period of about 1 to 4 minutes, 1 to 4 hours, or 1 to 4 weeks or longer, such as 5 to 20 weeks or longer, from the time of administration. In certain embodiments, the time period may be even longer. The dosage will be determined by the efficacy of the specific activating agent and the condition of the animal (e.g., human) and the weight of the animal (e.g., human) to be treated.

[0205] Many assays for determining the dosage to be administered are known in the art. For purposes herein, an assay can be used to determine the starting dose to be administered to a mammal, the assay comprising comparing the extent of treatment of cancer after administering a given dose of an agent of the present disclosure to one of a group of mammals, wherein each group of mammals is administered a different dose of the agent. The extent of treatment of cancer after administering a certain dose can be represented by the extent of tumor regression achieved, for example, with the agent in a mouse xenograft model. Methods for determining tumor regression are known in the art and are described herein.

[0206] The dosage of the active agent of the present invention will also be determined by the presence, nature and degree of any adverse side effects that may accompany the use of the specific active agent of the present invention. Generally, the attending physician will consider a variety of factors, such as age, body weight, general health, diet, sex, the active agent of the present invention to be administered, the route of administration and the severity of the disease being treated, to determine the dosage of the active agent of the present invention for treating each individual patient. For example, but not intended to limit the present disclosure, the dosage of the active agent of the present invention can be about 0.0001g to about 1g per kilogram of body weight of the treated subject per day, about 0.0001g to about 0.001g per kilogram of body weight per day, or about 0.01mg to about 1g per kilogram of body weight per day.

[0207] Controlled release formulations

[0208] In some embodiments, the active agent described herein can be modified into a reservoir form, whereby the active agent of the present disclosure is released into the body to which it is applied, and the mode is controlled according to the time and position in the body (see, e.g., U.S. Patent No. 4,450,150). The reservoir form of the active agent of the present disclosure can be, for example, an implantable composition comprising an active agent and a porous or non-porous material (e.g., a polymer), wherein the active agent is encapsulated by the material or diffused into the entire material and / or the non-porous material degrades. Then, the reservoir is implanted in the desired position in the subject's body, and the active agent is released from the implant at a predetermined rate.

[0209] In some aspects, the pharmaceutical composition comprising an activating agent is modified to have any type of in vivo release characteristics. In some aspects, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release or biphasic release formulation. Methods for preparing peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374:46-52 (2009) and International Patent Application Publication Nos. WO 2008 / 130158, WO2004 / 033036, WO2000 / 032218 and WO1999 / 040942, each of which is incorporated herein by reference.

[0210] The compositions of the invention may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in a prolonged release form to provide a prolonged storage and / or delivery effect.

[0211] use

[0212] The antigen binding proteins of the present disclosure can be used to inhibit tumor growth. Without being bound by a particular theory, the inhibitory effects of the antigen binding proteins provided herein allow such entities to be used in methods of treating cancer.

[0213] Thus, provided herein are methods of inhibiting tumor growth in a subject and methods of reducing tumor size in a subject. In various embodiments, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to inhibit tumor growth or reduce tumor size in the subject. In various aspects, the growth of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is inhibited. In various aspects, the size of an ovarian tumor, a melanoma tumor, a bladder tumor, or an endometrial tumor is reduced.

[0214] As used herein, the term "inhibit" or "reduce" and its derivatives may not be 100% or completely inhibited or reduced. In fact, there are different degrees of inhibition or reduction, and these inhibitions or reductions are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, the antigen-binding proteins of the present disclosure can inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of the present disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is at least or about a 10% reduction (e.g., at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about a 80% reduction, at least or about a 90% reduction, at least or about a 95% reduction, at least or about a 98% reduction).

[0215] Additionally provided herein are methods of treating a subject having cancer, such as a cancer expressing CDH17. In various embodiments, the method comprises administering to the subject an amount of a pharmaceutical composition of the present disclosure effective to treat the subject's cancer.

[0216] For purposes herein, the cancer in the methods disclosed herein can be any cancer, such as any malignant growth or tumor caused by abnormal and uncontrolled cell division, which can spread to other parts of the body through the lymphatic system or bloodstream. In some aspects, the cancer is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin's lymphoma (Hodgkin In certain aspects, the cancer is selected from the group consisting of: head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer, pancreatic cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), bronchioloalveolar carcinoma. In various aspects, the cancer is pancreatic cancer, gastrointestinal cancer, bladder cancer, colon cancer, lung cancer, liver cancer, endometrial cancer. In various aspects, the cancer is any cancer characterized by moderate to high expression of CDH17. See e.g. Figure 1 In various aspects, the cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, renal clear cell carcinoma, head and neck cancer, sarcoma, renal chromophobe cell carcinoma, low grade glioma, adrenocortical carcinoma, glioblastoma, renal papillary cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometrial cancer, uterine carcinosarcoma or ovarian cancer. In various aspects, the cancer is selected from pancreatic cancer, gastrointestinal cancer, bladder cancer, colon cancer, lung cancer, liver cancer, ovarian cancer, endometrioid carcinoma, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC) and cervical cancer.

[0217] As used herein, the term "treatment" and its related words do not necessarily mean 100% or complete treatment. In fact, there are different degrees of treatment, which are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, the method for treating cancer disclosed herein can provide any amount or any level of treatment. In addition, the treatment provided by the method disclosed herein can include the treatment of one or more diseases or symptoms or signs of the treated cancer. In addition, the treatment provided by the method disclosed herein can include slowing down the progress of cancer. For example, the method can treat cancer by enhancing T cell activity or immune response to cancer, reducing the growth of tumors or cancer, reducing the metastasis of tumor cells, increasing cell death of tumors or cancer cells, etc. In various aspects, the method is treated by delaying the onset or recurrence of cancer by at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years or longer. In various aspects, the method is treated by increasing the survival of the subject.

[0218] The antigen binding proteins of the present disclosure can also be used to detect CDH17 in a sample or diagnose CDH17-positive cancer. Therefore, the present disclosure provides a method for detecting CDH17 in a sample. In various embodiments, the method includes contacting the sample with an antigen binding protein, conjugate, or fusion protein as described herein, and determining an immune complex comprising an antigen binding protein, conjugate, or fusion protein that binds to CDH17. The present disclosure also provides a method for diagnosing CDH17-positive cancer in a subject. In various embodiments, the method includes contacting a biological sample comprising cells or tissues obtained from a subject with an antigen binding protein, conjugate, or fusion protein described herein, and determining an immune complex comprising an antigen binding protein, conjugate, or fusion protein that binds to CDH17.

[0219] Subjects

[0220] In some embodiments of the present disclosure, the subject is a mammal, including but not limited to rodents (order Rodentia) mammals, such as mice and hamsters; and lagomorphs (order Logomorpha) mammals, such as rabbits; carnivores (order Carnivora) mammals, including felines (cats) and canines (dogs); even-toed ungulates (order Artiodactyla) mammals, including bovines (cows) and suids (pigs); or odd-toed ungulates (order Perssodactyla) mammals, including equines (horses). In some aspects, the mammal belongs to the order Primates, Ceboids or Simoids (monkeys) or the order Anthropoids (humans and apes). In some aspects, the mammal is a human.

[0221] Pill Box

[0222] In some embodiments, the antigen-binding proteins of the present disclosure are provided in the form of a medicine kit. In various aspects, the medicine kit comprises one or more antigen-binding proteins in a unit dose. For the purposes of this article, "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, a unit dose is an amount sufficient to provide a desired effect to a subject, such as inhibiting tumor growth, reducing tumor size, and treating cancer. Therefore, a medicine kit is provided herein, which comprises the antigen-binding proteins of the present disclosure optionally provided in a unit dose. In various aspects, the medicine kit comprises several unit doses, such as a unit dose of a weekly or monthly supply, and optionally, each unit dose is packaged separately or otherwise separated from other unit doses. In some embodiments, the components of the medicine kit / unit dose are packaged together with instructions for administration to a patient. In some embodiments, the medicine kit comprises one or more devices for administration to a patient, such as a needle and a syringe, etc. In some aspects, the antigen-binding proteins of the present disclosure, pharmaceutically acceptable salts thereof, conjugates comprising antigen-binding proteins, or polymers or dimers comprising antigen-binding proteins are pre-packaged in a ready-to-use form, such as syringes, intravenous bags, etc. In some aspects, the kit also includes other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of the carriers described herein. In specific aspects, the kit includes an antigen binding protein of the present disclosure, and an agent for chemotherapy or radiotherapy, such as a therapeutic agent.

[0223] Various implementation plans

[0224] In various embodiments of the present disclosure, the antigen binding protein binds to the human CDH17 protein. In some embodiments, the antigen binding protein of the present disclosure binds to the ECD of CDH17. In some embodiments, the antigen binding protein binds to one or more peptides or polypeptides derived from the ECD of CDH17. In some embodiments, the binding of the antigen binding protein to CDH17 depends on the amino acid sequence of the peptide or polypeptide. In some aspects of the present invention, the antigen binding protein for CDH17 binds to the three-dimensional conformation (i.e., tertiary structure) of CDH17 or a peptide or polypeptide derived from the ECD of CDH17. In other embodiments, the antigen binding protein for CDH17 binds to the primary structure or conformation of CDH17 or a peptide or polypeptide (i.e., linear conformation) derived from the ECD of CDH17. In preferred embodiments, the preferred antigen binding protein for CDH17 binds to LDANGII derived from the ECD of CDH17. In other preferred embodiments, the antigen binding protein binds to DANGI derived from the ECD of CDH17. Specific examples of antigen-binding proteins that bind to CDH17 protein include, but are not limited to, the proteins shown in Tables 1-5 and 9.

[0225] In some embodiments, the antigen binding proteins of the present disclosure comprise an Fc polypeptide.In some embodiments, the antigen binding proteins of the present disclosure comprise an Fc polypeptide comprising afucosylated glycans.

[0226] In various aspects, the antigen binding proteins of the present disclosure are antibodies, such as monoclonal antibodies. In various cases, the antigen binding proteins are IgG. In various aspects, the antigen binding proteins inhibit at least about 50% colony growth in a soft agar 3D proliferation assay, or inhibit tumor growth in xenograft mice injected with human cancer cells. In various aspects, the antigen binding proteins inhibit tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various cases, the antigen binding proteins inhibit at least 50% tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0227] In certain embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 comprising the following amino acid sequence: GYTFXDXT (SEQ ID NO: 55), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F; (b) a heavy chain CDR2 comprising the following amino acid sequence: IFPRDDIV (SEQ ID NO: 14), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the following amino acid sequence: ARPP YYYSRNFYFDY (SEQ ID NO: 15), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the following amino acid sequence: SIISSSK (SEQ ID NO: 16), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (e) a light chain CDR2 comprising the following amino acid sequence: GTS (SEQ ID NO: 17). NO: 17), or a variant sequence thereof having only one or two amino acids different; (f) a light chain CDR3 comprising the following amino acid sequence: QQWSNYPFT (SEQ ID NO: 18), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). In some embodiments, the antigen binding protein also binds to an epitope comprising an amino acid sequence of any of the following: THNLQVAALDAN GIIVEGPVPIT (SEQ ID NO: 82), THNLQVAALDANGII (SEQ ID NO: 83), QVAALDANGIIVEGP (SEQ ID NO: 84), LDANGIIVEG PVPIT (SEQ ID NO: 85), LDANGII (SEQ ID NO: 53), or DANGI (SEQ ID NO: 54).

[0228] In certain embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 comprising the following amino acid sequence: GYTFXSXN (SEQ ID NO: 56), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F; (b) a heavy chain CDR2 comprising the following amino acid sequence: IYPGNGDT (SEQ ID NO: 2), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the following amino acid sequence: ARGRGRYFEY (SEQ ID NO: 3), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the following amino acid sequence: SSVSSSY (SEQ ID NO: 4), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (e) a light chain CDR2 comprising the following amino acid sequence: STS (SEQ ID NO: 57). NO:5), or a variant sequence thereof having only one or two amino acids different; (f) a light chain CDR3 comprising the following amino acid sequence: QQYDSSPST (SEQ ID NO:6), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). In some embodiments, the antigen binding protein also binds to an epitope comprising an amino acid sequence of any of the following: THNLQVAALDANGIIVEGPVPIT (SEQ ID NO:82), THNLQVAALDANGII (SEQ ID NO:83), QVAALDANGIIVEGP (SEQ ID NO:84), LDANGIIVEGPVPIT (SEQ ID NO:85), LDANGII (SEQ ID NO:53), or DANGI (SEQ ID NO:54).

[0229] In certain embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 comprising the following amino acid sequence: GYTFXDXY (SEQ ID NO: 57), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F; (b) a heavy chain CDR2 comprising the following amino acid sequence: IYPYSGGI (SEQ ID NO: 8), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the following amino acid sequence: ARGR GDYFGLFDF (SEQ ID NO: 9), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the following amino acid sequence: SSLSY (SEQ ID NO: 10), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (e) a light chain CDR2 comprising the following amino acid sequence: EIS (SEQ ID NO: 11). NO:11), or a variant sequence thereof having only one or two amino acids different; (f) a light chain CDR3 comprising the following amino acid sequence: QQWNYPFT (SEQ ID NO:12), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). In some embodiments, the antigen binding protein also binds to an epitope comprising an amino acid sequence of any of the following: THNLQVAALDANGIIVEG PVPIT (SEQ ID NO:82), THNLQVAALDANGII (SEQID NO:83), QVAALDANGIIVEGP (SEQ ID NO:84), LDANGIIVEGPVPIT (SEQ ID NO:85), LDANGII (SEQ ID NO:53), or DANGI (SEQ ID NO:54).

[0230] In certain embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 comprising the following amino acid sequence: GYTFXXXX (SEQ ID NO: 81), wherein X at position 5 is N, S, R, Q, A or T, X at position 6 is D or S, X at position 7 is H, W, Y or F, and X at position 8 is Y, T or N; (b) a heavy chain CDR2 comprising the following amino acid sequence: IFPRDDIV (SEQ ID NO: 14), or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the following amino acid sequence: ARPPYYYSR NFYFDY (SEQ ID NO: 15), or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; (d) a light chain CDR1 comprising the following amino acid sequence: SIISSSK (SEQ ID NO: 16). NO: 16), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (e) a light chain CDR2 comprising the following amino acid sequence: GTS (SEQ ID NO: 17), or a variant sequence thereof having only one or two amino acids different; (f) a light chain CDR3 comprising the following amino acid sequence: QQWSNYPFT (SEQ ID NO: 18), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). In some embodiments, the antigen binding protein also binds to an epitope comprising an amino acid sequence of any of the following: THNLQVAALDANGIIV EGPVPIT (SEQ ID NO: 82), THNLQVAALDANGII (SEQ ID NO: 83), QVAALDANGIIVEGP (SEQ ID NO: 84), LDANGIIVEGPVPI T (SEQ ID NO: 85), LDANGII (SEQ ID NO: 53), or DANGI (SEQ ID NO: 54).

[0231] In certain embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 comprising the following amino acid sequence: GYTFXXXX (SEQ ID NO: 81), wherein X at position 5 is N, S, R, Q, A or T, X at position 6 is D or S, X at position 7 is H, W, Y or F, and X at position 8 is Y, T or N; (b) a heavy chain CDR2 comprising the following amino acid sequence: IYPGNGDT (SEQ ID NO: 2), or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the following amino acid sequence: ARGRGRYFEY (SEQ ID NO: 3), or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; (d) a light chain CDR1 comprising the following amino acid sequence: SSVSSSY (SEQ ID NO: 4). NO:4), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (e) a light chain CDR2 comprising the following amino acid sequence: STS (SEQ ID NO:5), or a variant sequence thereof having only one or two amino acids different; (f) a light chain CDR3 comprising the following amino acid sequence: QQYDSSPST (SEQ ID NO:6), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). In some embodiments, the antigen binding protein also binds to an epitope comprising an amino acid sequence of any of the following: THNLQVAALDANGIIVEGPVPIT (SEQ ID NO:82), THNLQVAALDANGII (SEQ ID NO:83), QVAALDANGIIVEGP (SEQ ID NO:84), LDANGIIVEGPVPIT (SEQ ID NO:85), LDANGII (SEQ ID NO:53), or DANGI (SEQ ID NO:54).

[0232] In certain embodiments, the antigen binding protein comprises: (a) a heavy chain CDR1 comprising the following amino acid sequence: GYTFXXXX (SEQ ID NO: 81), wherein X at position 5 is N, S, R, Q, A or T, X at position 6 is D or S, X at position 7 is H, W, Y or F, and X at position 8 is Y, T or N; (b) a heavy chain CDR2 comprising the following amino acid sequence: IYPYSGGI (SEQ ID NO: 8), or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the following amino acid sequence: ARGRGDYFGLFDF (SEQ ID NO: 9), or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; (d) a light chain CDR1 comprising the following amino acid sequence: SSLSY (SEQ ID NO: 10). NO:10), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (e) a light chain CDR2 comprising the following amino acid sequence: EIS (SEQ ID NO:11), or a variant sequence thereof having only one or two amino acids different; (f) a light chain CDR3 comprising the following amino acid sequence: QQWNYPFT (SEQ ID NO:12), or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f). In some embodiments, the antigen binding protein also binds to an epitope comprising an amino acid sequence of any of the following: THNLQVAALDANGIIVEGPVPIT (SEQ ID NO:82), THNLQVAALDANGII (SEQ ID NO:83), QVAALDANGIIVEGP (SEQ ID NO:84), LDANGIIVEGPVPIT (SEQ ID NO:85), LDANGII (SEQ ID NO:53), or DANGI (SEQ ID NO:54).

[0233] In certain embodiments, the antigen binding protein specifically binds to human cadherin-17 (CDH17) at an epitope comprising the amino acid sequence of any of the following: THNLQVAALD ANGIIVEGPVPIT (SEQ ID NO:82), THNLQVAALDANGII (SEQ ID NO:83), QVAALDANGIIVEGP (SEQ ID NO:84), LDANGII VEGPVPIT (SEQ ID NO:85), LDANGII (SEQ ID NO:53), or DA NGI (SEQ ID NO:54).

[0234] In certain embodiments, the antigen binding protein specifically binds to human cadherin-17 (CDH17) at an epitope comprising the amino acid sequence of any of the following: THNLQVAALD ANGIIVEGPVPIT (SEQ ID NO: 82), THNLQVAALDANGII (SEQ ID NO: 83), QVAALDANGIIVEGP (SEQ ID NO: 84), LDANGII VEGPVPIT (SEQ ID NO: 85), LDANGII (SEQ ID NO: 53) or DA NGI (SEQ ID NO: 54), wherein binding of the antigen binding protein to the CDH17 epitope reduces or inhibits CDH17 activity.

[0235] In certain embodiments, the antigen binding protein specifically binds to human cadherin-17 (CDH17) at an epitope comprising the amino acid sequence of any of the following: THNLQVAALD ANGIIVEGPVPIT (SEQ ID NO:82), THNLQVAALDANGII (SEQ ID NO:83), QVAALDANGIIVEGP (SEQ ID NO:84), LDANGII VEGPVPIT (SEQ ID NO:85), LDANGII (SEQ ID NO:53) or DA NGI (SEQ ID NO:54), thereby inhibiting tumor growth, reducing tumor size, preventing cancer recurrence and / or treating cancer in a subject diagnosed as a CDH17 overexpressor.

[0236] The present disclosure provides a bispecific antigen-binding protein that binds to CDH17 and a second antigen, wherein the antigen-binding protein that binds to CDH17 is any one of the antigen-binding proteins described herein. In some embodiments, the bispecific antigen-binding protein comprises: (a) a heavy chain variable region amino acid sequence shown in Tables 1-4, or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; (b) a light chain variable region amino acid sequence shown in Tables 1-4, or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or (c) both (a) and (b). In some embodiments, the variant sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the bispecific antigen binding protein comprises an Fc polypeptide. In some embodiments, the bispecific antigen binding protein comprises an Fc polypeptide comprising afucosylated glycan.

[0237] In various aspects, the bispecific antigen binding protein binds CDH17 and a second antigen. In some embodiments, the bispecific antigen binding protein comprises an antigen binding fragment of an antibody specific for a second antigen. In various embodiments, the second antigen is a cell surface protein expressed by a T cell, optionally a component of a T cell receptor (TCR), such as CD3. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD3E. In some embodiments, the CDH17-CD3 bispecific antibody is 07-0653-h43Bs, 07-0646-h7Bs or 07-0663-h7Bs.

[0238] In various embodiments, the second antigen is a co-stimulatory molecule that contributes to T cell activation, such as CD40 or 4-1BB (CD137). In various embodiments, the second antigen is an Fc receptor, optionally, an Fcγ receptor, an Fc-α receptor or an Fc-ε receptor. In some embodiments, the Fc receptor is CD64 (Fc-γRI), CD32 (Fc-γRIIA), CD16A (Fc-γRIIIA), CD16b (Fc-γRIIIb), FcεRI, CD23 (Fc-εRII), CD89 (Fc-εRI), Fcα / μR, or FcRn. In some embodiments, the Fc receptor is CD16A.

[0239] In various embodiments, the second antigen is an immune checkpoint molecule, such as a protein involved in an immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA or SIGLEC7. In some embodiments, the immune checkpoint molecule is PD-1, LAG3, TIM3 or CTLA4. In various embodiments, the bispecific antigen binding protein comprises any scFv, Fab or F(ab)2' of any of the currently disclosed CDH17 antibodies.

[0240] In various embodiments, the bispecific antigen binding protein comprises an antigen binding protein comprising a sequence shown in Tables 1-4, or a variant sequence thereof having only 1-5 amino acids different or having at least or about 70% sequence identity. In some embodiments, the variant sequence has at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the bispecific antigen binding protein comprises a nanobody, a diabody, The structure of DART, TandAb, CrossMab or HSAbody.

[0241] The present disclosure provides a conjugate comprising an antigen binding protein or a bispecific antigen binding protein described herein and a heterologous portion. In some embodiments, the antigen binding protein comprises the amino acid sequence shown in Tables 1-4. In some embodiments, the conjugate comprises a cytotoxic agent or a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization. In some embodiments, the antimitotic agent is auristatin. In some embodiments, auristatin is MMAE.

[0242] In various embodiments, the conjugates of the present disclosure are conjugated to the antigen binding protein via a cleavable linker. In some embodiments, the cleavable linker is VC-PAB (eg, MC-VC-PAB).

[0243] In some embodiments, the conjugate comprises an antigen binding protein, the antigen binding protein is an antibody, the antibody is a monoclonal antibody, optionally wherein the monoclonal antibody is an IgG antibody. In some embodiments, the antibody is a human antibody, a humanized antibody or a chimeric antibody.

[0244] In various embodiments, the conjugates of the present disclosure have an average unit number of agents conjugated to each antigen-binding protein in the range of 1 to 8, preferably wherein the average unit number of agents conjugated to each antigen-binding protein is in the range of 3-8. In some embodiments, the conjugate is a heterogeneous conjugate. In other embodiments, the conjugate is a homogeneous conjugate. In some embodiments, the conjugate comprises a heterologous portion or agent, wherein the agent is conjugated to a specific site of the antigen-binding protein. In some embodiments, the specific site is an unpaired cysteine ​​residue.

[0245] The present disclosure also provides a fusion protein, the fusion protein comprises an antigen binding protein or a bispecific antigen binding protein described herein. The present disclosure also provides a nucleic acid, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein, a bispecific antigen binding protein, a conjugate or a fusion protein of the present disclosure. The present disclosure provides a vector, the vector comprises a nucleic acid, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein, a conjugate or a fusion protein of the present disclosure. The present disclosure further provides a host cell comprising a nucleic acid or a vector of the present disclosure.

[0246] The present disclosure provides a method for producing an antigen binding protein or a bispecific antigen binding protein that binds to a CDH17 protein, the method comprising (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein or a bispecific antigen binding protein described herein, and (ii) harvesting the antigen binding protein or the bispecific antigen binding protein from the cell culture medium. In addition, a method for producing a fusion protein is provided, the fusion protein comprising an antigen binding protein or a bispecific antigen binding protein that binds to a CDH17 protein, the method comprising (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding a fusion protein of the present disclosure, and (ii) harvesting the fusion protein from the cell culture medium.

[0247] The present disclosure also provides a method for producing a pharmaceutical composition, the method comprising combining the antigen-binding protein, bispecific antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell or a combination thereof disclosed herein with a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition is also provided, the pharmaceutical composition comprising the antigen-binding protein, bispecific antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, host cell or a combination thereof disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0248] Provided herein is a method for treating a subject with a cancer expressing CDH17, the method comprising administering to the subject an amount of a pharmaceutical composition described herein effective to treat the cancer. Also provided is a method for inhibiting tumor growth in a subject, the method comprising administering to the subject an amount of a pharmaceutical composition described herein effective to inhibit tumor growth. The present disclosure provides a method for reducing tumor size in a subject, the method comprising administering to the subject an amount of a pharmaceutical composition described herein effective to reduce tumor size. Also provided is a method for preventing cancer recurrence in a subject, the method comprising administering to the subject an amount of a pharmaceutical composition described herein effective to prevent cancer recurrence.

[0249] The present disclosure provides a method for detecting CDH17 in a sample, the method comprising contacting the sample with an antigen binding protein, bispecific antigen binding protein, conjugate or fusion protein of the present disclosure, and determining an immune complex comprising the antigen binding protein, conjugate or fusion protein that binds to CDH17. Also provided herein is a method for diagnosing a CDH17-positive cancer in a subject, the method comprising contacting a biological sample comprising cells or tissues obtained from the subject with an antigen binding protein, bispecific antigen binding protein, conjugate or fusion protein of the present disclosure, and determining an immune complex comprising the antigen binding protein, conjugate or fusion protein that binds to CDH17.

[0250] The present disclosure also provides a method for treating cancer in a subject diagnosed as a low overexpressor of CDH17. In various embodiments, the method comprises administering to the subject a pharmaceutical composition disclosed herein in an amount effective to prevent cancer recurrence. In some aspects, the administration induces apoptosis of tumor cells, and optionally, the administration induces apoptosis of cells expressing CDH17. In various aspects, the subject has a tumor and the tumor is semi-quantitatively classified into one of four groups: high expressers, moderate expressers, low expressers, and non-expressers.

[0251] The present disclosure also provides a method for producing a conjugate. In some embodiments, the method includes contacting the antigen-binding protein or its fusion protein with an agent comprising a chemically reactive group, and the chemically reactive group can react with any part of the antigen-binding protein or its fusion protein. In some embodiments, the method includes contacting the antigen-binding protein or its fusion protein with an agent and a bifunctional linker, and the bifunctional linker can bridge the linker and the antigen-binding protein or its fusion protein. In some embodiments, the agent is a cytotoxic agent and / or a chemotherapeutic agent.

[0252] In some embodiments, the method of producing a conjugate comprises (a) culturing host cells in a cell culture medium, wherein the host cells contain a nucleotide sequence encoding an antigen binding protein or a fusion protein thereof; (b) harvesting the antigen binding protein or the fusion protein from the cell culture medium; and (c) linking the antigen binding protein or the fusion protein to a second moiety to produce the conjugate, wherein the second moiety is a cytotoxic agent or a chemotherapeutic agent.

[0253] Exemplary embodiments

[0254] 1. An antigen binding protein, comprising:

[0255] a. CDRs 1-3 derived from a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQG LEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSE DTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSS (SEQ ID NO: 49) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity; and / or

[0256] b. CDRs 1-3 derived from a light chain variable region comprising the following amino acid sequence:

[0257] DIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGK APKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQ QWSNYPFTFGQGTKLEIK (SEQ ID NO: 50) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity.

[0258] 2. An antigen-binding protein, comprising:

[0259] a. CDRs 1-3 derived from a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTSYNMHWVRQAPG QGLEWIGAIYPGNGDTSYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGRGRYFEYWGQGTTLTVSS (SEQ ID NO: 45) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity; and / or

[0260] b. CDRs 1-3 derived from a light chain variable region comprising the following amino acid sequence:

[0261] DIQLTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKPG KAPKLLIYSTSNLASGVPSRFSGSGSGTDYTLTISSVQPEDFATYYC QQYDSSPSTFGQGTKLEIK (SEQ ID NO: 46) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity.

[0262] 3. An antigen binding protein, comprising:

[0263] a. CDRs 1-3 derived from a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPG QGLEWMGVIYPYSGGIGYAQKFQGRVTMTVDKSTSTAYMELSSL RSEDTAVYYCARGRGDYFGLFDFWGQGTTVTVSS (SEQ ID NO: 47) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity; and / or

[0264] b. CDRs 1-3 derived from a light chain variable region comprising the following amino acid sequence:

[0265] DIQLTQSPSSLSASVGDRVTITCRATSSLSYIHWYQQKPGKAP KPLIYEISKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWNYPFTFGQGTKLEIK (SEQ ID NO: 48) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity.

[0266] 4. An antigen binding protein, comprising:

[0267] a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFTDHT (SEQ ID NO: 13) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0268] b. a heavy chain CDR2 comprising the following amino acid sequence: IFPRDDIV (SEQ ID NO: 14) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0269] c. a heavy chain CDR3 comprising the following amino acid sequence: ARPPYYYSRNFYFDY (SEQ ID NO: 15) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0270] d. a light chain CDR1 comprising the following amino acid sequence: SIISSSK (SEQ ID NO: 16) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0271] e. a light chain CDR2 comprising the following amino acid sequence: GTS (SEQ ID NO: 17) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0272] f. a light chain CDR3 comprising the following amino acid sequence: QQWSNYPFT (SEQ ID NO: 18) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or

[0273] g. A combination of any two or more of (a)-(f).

[0274] 5. An antigen binding protein, comprising:

[0275] a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFTSYN (SEQ ID NO: 1) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0276] b. a heavy chain CDR2 comprising the following amino acid sequence: IYPGNGDT (SEQ ID NO: 2) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0277] c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGRYFEY (SEQ ID NO: 3) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0278] d. a light chain CDR1 comprising the following amino acid sequence: SSVSSSY (SEQ ID NO: 4) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0279] e. a light chain CDR2 comprising the following amino acid sequence: STS (SEQ ID NO: 5) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0280] f. a light chain CDR3 comprising the following amino acid sequence: QQYDSSPST (SEQ ID NO: 6) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or

[0281] g. A combination of any two or more of (a)-(f).

[0282] 6. An antigen binding protein, comprising:

[0283] a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFTDYY (SEQ ID NO: 7) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0284] b. a heavy chain CDR2 comprising the following amino acid sequence: IYPYSGGI (SEQ ID NO: 8) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0285] c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGDYFGLFDF (SEQ ID NO: 9) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0286] d. a light chain CDR1 comprising the following amino acid sequence: SSLSY (SEQ ID NO: 10) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0287] e. a light chain CDR2 comprising the following amino acid sequence: EIS (SEQ ID NO: 11) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0288] f. a light chain CDR3 comprising the following amino acid sequence: QQWNYPFT (SEQ ID NO: 12) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or

[0289] g. A combination of any two or more of (a)-(f).

[0290] 7. The antigen-binding proteins according to claim 4, further comprising:

[0291] a. a heavy chain FR1 comprising the following amino acid sequence: QVQLVQSGAEVKKPGS SVKISCKVS (SEQ ID NO: 37) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0292] b. a heavy chain FR2 comprising the following amino acid sequence: IHWMRQAPGQGLEWI GY (SEQ ID NO: 38) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0293] c. a heavy chain FR3 comprising the following amino acid sequence: VYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 39) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0294] d. a heavy chain FR4 comprising the following amino acid sequence: WGQGTTLTVSS (SEQ ID NO: 40) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0295] e. a light chain FR1 comprising the following amino acid sequence: DIQMTQSPSSLSASVGD RVTITCRVS (SEQ ID NO: 41) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0296] f. a light chain FR2 comprising the following amino acid sequence: LHWYQQKPGKAPKPLIY (SEQ ID NO: 42) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0297] g. a light chain FR3 comprising the following amino acid sequence: TLASGVPSRFSGSGSGT DYTLTISSLQPEDFATYYC (SEQ ID NO: 43) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0298] h. a light chain FR4 comprising the following amino acid sequence: FGQGTKLEIK (SEQ ID NO: 44) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or

[0299] i. A combination of any two or more of (a)-(h).

[0300] 8. The antigen-binding proteins of 5, further comprising:

[0301] a. a heavy chain FR1 comprising the following amino acid sequence: QVQLVQSGAEVKKPG ASVKMSCKAS (SEQ ID NO: 21) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0302] b. a heavy chain FR2 comprising the following amino acid sequence: MHWVRQAPGQGLEWI GA (SEQ ID NO: 22) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0303] c. a heavy chain FR3 comprising the following amino acid sequence: SYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 23) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0304] d. a heavy chain FR4 comprising the following amino acid sequence: WGQGTTLTVSS (SEQ ID NO: 24) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0305] e. a light chain FR1 comprising the following amino acid sequence: DIQLTQSPSSLSASVGD RVTMTCRAS (SEQ ID NO: 25) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0306] f. a light chain FR2 comprising the following amino acid sequence: LHWYQQKPGKAPKLLIY (SEQ ID NO: 26) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0307] g. a light chain FR3 comprising the following amino acid sequence: NLASGVPSRFSGSGSG TDYTLTISSVQPEDFATYYC (SEQ ID NO: 27) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0308] h. a light chain FR4 comprising the following amino acid sequence: FGQGTKLEIK (SEQ ID NO: 28) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or

[0309] i. A combination of any two or more of (a)-(h).

[0310] 9. The antigen-binding proteins according to claim 6, further comprising:

[0311] a. a heavy chain FR1 comprising the following amino acid sequence: QVQLVQSGAEVKKPG ASVKVSCKAS (SEQ ID NO: 29) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0312] b. a heavy chain FR2 comprising the following amino acid sequence: MNWVRQAPGQGLEW MGV (SEQ ID NO: 30) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0313] c. a heavy chain FR3 comprising the following amino acid sequence: GYAQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 31) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0314] d. a heavy chain FR4 comprising the following amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 32) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0315] e. a light chain FR1 comprising the following amino acid sequence: DIQLTQSPSSLSASVGD RVTITCRAT (SEQ ID NO: 33) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0316] f. a light chain FR2 comprising the following amino acid sequence: IHWYQQKPGKAPKPLIY (SEQ ID NO: 34) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity;

[0317] g. a light chain FR3 comprising the following amino acid sequence: KLASGVPSRFSGSGSG TDYTLTISSLQPEDFATYYC (SEQ ID NO: 35) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity;

[0318] h. a light chain FR4 comprising the following amino acid sequence: FGQGTKLEIK (SEQ ID NO: 36) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or

[0319] i. A combination of any two or more of (a)-(h).

[0320] 10. An antigen binding protein, comprising:

[0321] a. a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYA QKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSS (SEQ ID NO: 49) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0322] b. A light chain variable region comprising the following amino acid sequence:

[0323] DIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGK APKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQ QWSNYPFTFGQGTKLEIK (SEQ ID NO: 50) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0324] 11. An antigen binding protein, comprising:

[0325] a. a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPG ASVKMSCKASGYTFTSYNMHWVRQAPGQGLEWIGAIYPGNGDT SYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGRGRYFEYWGQGTTLTVSS (SEQ ID NO: 45) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0326] b. A light chain variable region comprising the following amino acid sequence:

[0327] DIQLTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKPG KAPKLLIYSTSNLASGVPSRFSGSGSGTDYTLTISSVQPEDFATYYC QQYDSSPSTFGQGTKLEIK (SEQ ID NO: 46) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0328] 12. An antigen binding protein, comprising:

[0329] a. a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPG ASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGVIYPYSGGI GYAQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGRGDYFGLFDFWGQGTTVTVSS (SEQ ID NO: 47) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0330] b. A light chain variable region comprising the following amino acid sequence:

[0331] DIQLTQSPSSLSASVGDRVTITCRATSSLSYIHWYQQKPGKAP KPLIYEISKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWNYPFTFGQGTKLEIK (SEQ ID NO:48) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0332] 13. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising:

[0333] a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFXDXT (SEQ ID NO: 55), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F;

[0334] b. a heavy chain CDR2 comprising the following amino acid sequence: IFPRDDIV (SEQ ID NO: 14) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0335] c. a heavy chain CDR3 comprising the following amino acid sequence: ARPPYYYSRNFYFDY (SEQ ID NO: 15) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0336] d. a light chain CDR1 comprising the following amino acid sequence: SIISSSK (SEQ ID NO: 16) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0337] e. a light chain CDR2 comprising the following amino acid sequence: GTS (SEQ ID NO: 17) or a variant sequence thereof that differs in only one or two amino acids;

[0338] f. a light chain CDR3 comprising the following amino acid sequence: QQWSNYPFT (SEQ ID NO: 18) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or

[0339] g. A combination of any two or more of (a)-(f).

[0340] 14. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising:

[0341] a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFXSXN (SEQ ID NO: 56), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F;

[0342] b. a heavy chain CDR2 comprising the following amino acid sequence: IYPGNGDT (SEQ ID NO: 2) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0343] c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGRYFEY (SEQ ID NO: 3) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0344] d. a light chain CDR1 comprising the following amino acid sequence: SSVSSSY (SEQ ID NO: 4) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0345] e. a light chain CDR2 comprising the following amino acid sequence: STS (SEQ ID NO: 5) or a variant sequence thereof that differs in only one or two amino acids;

[0346] f. a light chain CDR3 comprising the following amino acid sequence: QQYDSSPST (SEQ ID NO: 6) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or

[0347] g. A combination of any two or more of (a)-(f).

[0348] 15. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising:

[0349] a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFXDXY (SEQ ID NO: 57), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F;

[0350] b. a heavy chain CDR2 comprising the following amino acid sequence: IYPYSGGI (SEQ ID NO: 8) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0351] c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGDYFGLFDF (SEQ ID NO: 9) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity;

[0352] d. a light chain CDR1 comprising the following amino acid sequence: SSLSY (SEQ ID NO: 10) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity;

[0353] e. a light chain CDR2 comprising the following amino acid sequence: EIS (SEQ ID NO: 11) or a variant sequence thereof that differs in only one or two amino acids;

[0354] f. a light chain CDR3 comprising the following amino acid sequence: QQWNYPFT (SEQ ID NO: 12) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or

[0355] g. A combination of any two or more of (a)-(f).

[0356] 16. An antigen binding protein as described in any of the above or as described herein, wherein the variant sequence has at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

[0357] 17. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising:

[0358] a. Antibody heavy chain, containing the following amino acid sequence: QVQLVQSGAEVKKPGSS VKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:94); and

[0359] b. an antibody light chain comprising the following amino acid sequence: DIQMTQSPSSLSASVGDR VTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95).

[0360] 18. An antigen binding protein as described in any one of the above or as described herein, wherein:

[0361] a. The antigen binding protein binds to human cadherin-17 (CDH17) protein (SEQ ID NO: 19);

[0362] b. The antigen binding protein binds to the extracellular domain of human CDH17 with a dissociation constant (K D ) is less than about 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM;

[0363] c. The antigen binding protein preferentially binds to human CDH17 relative to mouse CDH17 (SEQ ID NO: 20);

[0364] d. the antigen binding protein does not bind to mouse CDH17; or

[0365] e. Combinations thereof.

[0366] 19. An antigen binding protein as described in any one of the above or as described herein, which specifically binds to human cadherin-17 (CDH17) or a polypeptide comprising the amino acid sequence of any one of the following: THNLQVAALDANGIIVEG PVPIT (SEQ ID NO: 82), THNLQVAALDANGII (SEQ ID NO: 83), QVAALDANGIIVEGP (SEQ ID NO: 84), LDANGIIVEGPVPIT (SEQ ID NO: 85), LDANGII (SEQ ID NO: 53) and DANGI (SEQ ID NO: 54).

[0367] 20. The antigen binding protein as described in any one of the above or as described herein, wherein the binding of the antigen binding protein to CDH17 reduces, interferes with or inhibits CDH17 activity.

[0368] 21. An antigen binding protein as described in any one of the above or as described herein, which is an antibody or an antibody fragment that binds to an antigen.

[0369] 22. The antigen-binding protein of 21, wherein the antibody is a monoclonal antibody.

[0370] 23. The antigen-binding protein of 21 or 22, wherein the antibody is a chimeric antibody, a human antibody or a humanized antibody.

[0371] 24. The antigen-binding protein of any one of 21-23, wherein the antibody is IgG.

[0372] 25. The antigen-binding protein of 24, wherein the IgG is selected from IgG1, IgG2, IgG3 and IgG4.

[0373] 26. An antigen binding protein as described in any one of the above or as described herein, wherein the antigen binding protein is a bispecific antigen binding protein or a bispecific T cell engager (BiTE).

[0374] 27. The antigen-binding protein of 26, wherein the bispecific antigen-binding protein or the BiTE comprises the amino acid sequence shown in:

[0375] a.SEQ ID NO:45 and SEQ ID NO:46;

[0376] b. SEQ ID NO:47 and SEQ ID NO:48; or

[0377] c. SEQ ID NO:49 and SEQ ID NO:50,

[0378] Optionally, the bispecific antigen binding protein is selected from 07-0653-h43Bs, 07-0646-h7Bs and 07-0663-h7Bs.

[0379] 28. The antigen-binding protein of 21, wherein the antigen-binding antibody fragment is selected from scFv, F(ab')2, Fab, Fab' and Fv.

[0380] 29. The antigen binding protein of 28, wherein the scFv is 07-0653-h43scfv, 07-0646-h7scfv or 07-0663-h7scfv.

[0381] 30. An antigen binding protein as described in any one of the above or as described herein, which inhibits tumor growth in xenograft mice injected with human cancer cells.

[0382] 31. An antigen binding protein as described in any one of the above or as described herein, comprising an Fc polypeptide comprising afucosylated glycans.

[0383] 32. A conjugate comprising the antigen binding protein described in any one of the above or as described herein or as described herein.

[0384] 33. The conjugate of 32, comprising a detectable label, a cytotoxic agent or a chemotherapeutic agent.

[0385] 34. The conjugate of 33, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking tubulin polymerization.

[0386] 35. The conjugate of 34, wherein the antimitotic agent is auristatin.

[0387] 36. The conjugate of 35, wherein the auristatin is MMAE.

[0388] 37. The conjugate of any one of 33-36, wherein the agent or the label is conjugated to the antigen binding protein via a cleavable linker or a non-cleavable linker.

[0389] 38. The conjugate of 37, wherein the cleavable linker is VC-PAB.

[0390] 39. The conjugate of any one of 32-38, wherein the antigen binding protein is an antibody.

[0391] 40. The immunoconjugate of 39, wherein the antibody is a monoclonal antibody.

[0392] 41. The conjugate of 39 or 40, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

[0393] 42. The conjugate of any one of 39-41, wherein the antibody is an IgG antibody, optionally wherein the IgG is IgG1, IgG2, IgG3 or IgG4.

[0394] 43. A conjugate as described in any of 32-42, wherein the average number of units of the agent conjugated to each antigen binding protein is in the range of 1 to 8, preferably wherein the average number of units of the agent conjugated to each antigen binding protein is (a) in the range of 3-8, or (b) 4.

[0395] 44. The conjugate of any one of 32-43, wherein the conjugate is a heteroconjugate.

[0396] 45. The conjugate of any one of 32-43, wherein the conjugate is a homogeneous conjugate.

[0397] 46. ​​The conjugate of any one of 32-45, wherein the agent is conjugated at a specific site of the antigen binding protein.

[0398] 47. The conjugate of 46, wherein the specific site is an unpaired cysteine ​​residue.

[0399] 48. The conjugate of any one of 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 45 and SEQ ID NO: 46.

[0400] 49. The conjugate of any one of 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 47 and SEQ ID NO: 48.

[0401] 50. The conjugate of any one of 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 49 and SEQ ID NO: 50.

[0402] 51. The conjugate of any one of 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 94 and SEQ ID NO: 95.

[0403] 52. A fusion protein comprising an antigen binding protein as described in any one of the above or as described herein.

[0404] 53. A nucleic acid comprising a nucleotide sequence encoding an antigen binding protein as described in any one of 1-31, a conjugate as described in 32-51, or a fusion protein as described in 52.

[0405] 54. The nucleic acid of 53, wherein the nucleic acid is cDNA.

[0406] 55. A vector (eg, an expression vector) comprising the nucleic acid of 53 or 54.

[0407] 56. The vector of 55, further comprising an internal ribosome entry site (IRES).

[0408] 57. A host cell comprising the nucleic acid of 53 or 54, or the vector of 55 or 56.

[0409] 58. The host cell of 57, wherein the host cell is a bacterial cell.

[0410] 59. The host cell of 57, wherein the host cell is a eukaryotic cell.

[0411] 60. The host cell of 59, wherein the eukaryotic cell is a mammalian cell.

[0412] 61. A host cell as described in 60, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

[0413] 62. A method for producing an antigen binding protein that binds to a Cadherin-17 (CDH17) protein, the method comprising (i) culturing the host cell of any one of 57-61 in a cell culture medium, and (ii) harvesting the antigen binding protein from the cell culture medium.

[0414] 63. A method for producing a fusion protein, wherein the fusion protein comprises an antigen binding protein that binds to a Cadherin-17 (CDH17) protein, the method comprising (i) culturing a host cell as described in any one of 57-61 in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.

[0415] 64. A method for producing a pharmaceutical composition, the method comprising combining: (a) an antigen binding protein as described in any one of 1-31, a conjugate as described in any one of 32-51, a fusion protein as described in 52, a nucleic acid as described in 53 or 54, a vector as described in 55 or 56, a host cell as described in 57, 59 or 60, or any combination thereof; and (b) a pharmaceutically acceptable carrier, diluent and / or excipient.

[0416] 65. A pharmaceutical composition comprising (a) an antigen binding protein as described in any one of 1-31, a conjugate as described in any one of 32-51, a fusion protein as described in 52, a nucleic acid as described in 53 or 54, a vector as described in 55 or 56, a host cell as described in 57, 59 or 60, or any combination thereof; and (b) a pharmaceutically acceptable carrier, diluent and / or excipient.

[0417] 66. A method of treating a subject having a cancer expressing CDH17, the method comprising administering to the subject an amount of the pharmaceutical composition of 65 effective to treat the cancer.

[0418] 67. A method for inhibiting tumor growth in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of 65 effective to inhibit tumor growth.

[0419] 68. A method of reducing tumor size in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of 65 effective to reduce tumor size.

[0420] 69. A method for preventing cancer recurrence in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of 65 effective to prevent the cancer recurrence.

[0421] 70. A method of treating cancer in a subject diagnosed as a CDH17 overexpresser, the method comprising administering to the subject an amount of the pharmaceutical composition according to 65 effective to prevent recurrence of the cancer.

[0422] 71. The method of any one of 66-70, wherein the administration induces apoptosis of tumor cells.

[0423] 72. The method of any one of 66-70, wherein the administration induces apoptosis of cells expressing CDH17.

[0424] 73. A method for detecting E-cadherin-17 (CDH17) in a sample, the method comprising contacting the sample with an antigen binding protein as described in any one of 1-31, a conjugate as described in any one of 32-51, or a fusion protein as described in 52; and determining an immune complex comprising the antigen binding protein, the conjugate, or the fusion protein bound to CDH17.

[0425] 74. A method for diagnosing a subject's Cadherin-17 (CDH17)-positive cancer, the method comprising contacting a biological sample comprising cells or tissues obtained from the subject with an antigen binding protein as described in any one of 1-31, a conjugate as described in any one of 32-51, or a fusion protein as described in 52; and determining an immune complex comprising the antigen binding protein, the conjugate, or the fusion protein bound to CDH17.

[0426] 75. The method of 74, further comprising treating the subject by administering to the subject diagnosed with a CDH17-positive cancer the antigen binding protein of any one of 1-31, the conjugate of any one of 32-51, or the fusion protein of 52.

[0427] 76. A method of producing a conjugate as described in any one of the above or as described herein, the method comprising

[0428] a. culturing a host cell as described in any one of 57-61 in a cell culture medium, wherein the host cell comprises a nucleic acid encoding an antigen binding protein or a fusion protein thereof,

[0429] b. harvesting the antigen binding protein or the fusion protein from the cell culture medium, and

[0430] c. linking the antigen binding protein or fusion protein to a heterologous moiety, thereby producing the conjugate, optionally wherein the heterologous moiety is a cytotoxic agent or a chemotherapeutic agent.

[0431] 77. A method for activating T cells so that they target tumors or cancer cells expressing CDH17 in a subject, the method comprising administering to the subject a bispecific T cell engager (BiTE), the BiTE comprising a first scFv that binds CDH17 and a second scFv that binds CD3, wherein the first scFv that binds CDH17 comprises the VH region and VL region of an antigen binding protein as described in any one of the above or as described herein.

[0432] 78. The method of 77, wherein the BiTE comprises an amino acid sequence as shown below:

[0433] a.SEQ ID NO:45 and SEQ ID NO:46;

[0434] b. SEQ ID NO:47 and SEQ ID NO:48; or

[0435] c. SEQ ID NO:49 and SEQ ID NO:50.

[0436] 79. The method of 77 or 78, wherein the BiTE is selected from 07-0646-h7Bs, 07-0653-h43Bs and 07-0663-h7Bs, optionally wherein the BiTe is 07-0653-h43Bs.

[0437] 80. A method for inducing an antibody-dependent cell-mediated cytotoxicity (ADCC) response against a tumor or cancer cell expressing CDH17 in a subject, the method comprising administering to the subject an antigen binding protein that binds CDH17, wherein the antigen binding protein comprises an Fc effector function; and a VH region and a VL region of an antigen binding protein as described in any one of the above or as described herein.

[0438] 81. The method of 80, wherein the antigen binding protein that binds to CDH17 is 07-646-h7 or 07-663-h7.

[0439] 82. The method of any one of the foregoing or as described herein, wherein the subject is a mammal, optionally a dog, cat, mouse or human.

[0440] The following examples are provided merely to illustrate the present disclosure and are not intended to limit the scope thereof in any way.

[0441] Example

[0442] Example 1

[0443] This example describes the generation of CDH17-specific antibodies.

[0444] Mice were immunized with 3T3 cells overexpressing epitope-tagged full-length human CDH17 protein using a mammalian expression vector encoding a human CDH17 fusion protein.

[0445] Splenocytes were harvested from immunized mice and fused to myeloma lines by BTX electrofusion (BTX, Holliston, MA) to generate hybridomas. 7000 primary hybridoma cultures were generated and cultured in 384-well plates. The ability of the antibodies to bind to the peptide and / or human cancer cells expressing CDH17 was assessed by ELISA assays and / or flow cytometry. Approximately 2000 potential positive antibodies were rearranged into 96-well plates and screened by flow cytometry against endogenous and artificial cell line models.

[0446] Hybridomas that are positive for producing antibodies that bind to human CDH17 protein are identified, and nucleic acid sequences encoding immunoglobulin light and heavy chains against human CDH17 protein are isolated. Using these nucleic acid sequences, ExpiCHO TMThe CDH17 antibody is expressed in the form of a full-length human IgG antibody (e.g., IgG1). The heavy and light chain variable regions of the antibody are cloned into an antibody expression vector and then cloned into a vector based on pcDNA3. TM The vector was engineered using the ExpiCHO TM The antibody expression vector was transfected into CHO cells using the protocol provided in the CDH17 expression system (Cat. No. A29133, ThermoFisher Scientific, USA) to produce bicistronic mRNA, in which the IRES drives the expression of the second immunoglobulin chain. It is noteworthy that the C-terminal lysine of the human IgG1 constant region in the chimeric CDH17 antibody was removed, but the C-terminal lysine of the humanized CDH17 antibody was not removed. Both the chimeric and humanized antibodies contain human IgG1 constant regions and kappa constant regions. The resulting antibodies were purified using protein A / G resin. Cell surface binding of the antibody to CDH17 was determined by FACS, in which the CDH17 antibody was directly stained with Alexa Fluor 485 antibody following the manufacturer's protocol. The antibody was conjugated with 647NHS ester (succinimidyl ester, catalog number A20106 (ThermoFisherScientific)). The binding of the antibody to CDH17 was also determined and its EC50 was measured using a recombinant CDH17 extracellular domain (ECD) (SEQ ID NO: 52) with a 6×His tag.

[0447] CDH17 expressing cells were used in FACS assays to determine the ability of CDH17 antibodies to bind CDH17 on the cell surface and to cross-react with other CDH17 family members. HEK293 T cells engineered to express human, monkey, mouse or rat CDH17 fused to fluorescent protein (mGFP or moxGFP) were used as artificial models of CDH17 expression. SNU-C1 (human colorectal cancer cell line), PaTu8988s (human pancreatic cancer cell line), HPAF-II (human pancreatic cancer cell line) and LS513 (human colorectal cancer cell line) were used as endogenous models of CDH17 expression, while H524 (human small cell lung cancer cell line), COR-L279 (human small cell lung cancer cell line) and M202 (human melanoma cell line) were used as endogenous models of CDH17 expression loss.

[0448] For each cell type and each mAb tested, cells were detached from the flask surface using versene (rather than trypsin) to protect cell surface proteins. The detached cells were then incubated with a predetermined concentration of Alexa Fluor The CDH17 mAb was directly labeled with Alexa Fluor 647NHS ester (succinimidyl ester). Alternatively, unlabeled CDH17 antibody can be detected using a fluorescently labeled secondary antibody. After washing, the CDH17 antibody was detected by BD Accuri TM Cells were read on a flow cytometer C6 to detect antibody-antigen protein binding in channel FL4H. Each antibody was tested at different concentrations to establish a dose-fluorescence curve.

[0449] Example 2

[0450] This example demonstrates the humanization of the antibodies of the present disclosure.

[0451] Selection of antibodies for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of mouse monoclonal anti-CDH17 antibodies were aligned with human V H Genes and People V L A library of known human germline sequences of the κ gene ( theinternational ImMunoGeneTics information www.imgt.org; founder and director: Marie-Paule Lefranc, Montpellier, France); the database used is the IMGT Human V H Gene (F+ORF, 273 germline sequences) and IMGT human V L Kappa gene (F+ORF, 74 germline sequences). The acceptor human germline was selected from those with the closest sequence to the parent antibody.

[0452] It may be necessary to change the human germline framework (ie, non-CDR residues in VH and VL; abbreviated as FR) positions to the corresponding parental murine sequences to optimize the binding of the humanized antibody. The sequences of various versions of humanized antibodies are provided in the figure.

[0453] The construction and expression of humanized antibodies CDH17-646-h7, CDH17-653-h42, CDH17-653-h43, CDH17-657-h16, CDH17-663-h7, CDH17-670-h12, CDH17-675-h11 and CDH17-683-h6 were essentially as described in Example 1. FACS assays were performed to determine the relative antigen binding strength of humanized antibodies (1 μg) binding to CDH17 overexpressed in HEK293T cell lines. The corresponding parental antibody (antibody before humanization) was used as a control and indicated with "chim".

[0454] Based on the in vitro antigen binding data, three humanized antibodies (07-0646-h7 (also known as CDH17-646-h7), 07-0653-h43 (also known as CDH17-653-h43), and 07-0663-h7 (also known as CDH17-663-h7)) were selected for further testing and development. These antibodies are derived from CDH17-646 (also known as CDH17-646-m), CDH17-653 (also known as CDH17-653-m), and CDH17-663.

[0455] In vivo binding studies of humanized versions of chimeric monoclonal anti-CDH17 antibodies CDH17-646, CDH 17-653, CDH 17-657, CDH 17-663, CDH 17-670, CDH 17-675, and CDH 17-683, as well as chimeric monoclonal anti-CDH17 antibodies and antibody-drug conjugates (ADC; antibody-MMAE conjugates) were performed in xenograft mice injected with CDH17-positive human colorectal cancer cell lines SNU-C1 and LS513, CDH17-positive human pancreatic cancer cell lines HPAF-II and PaTu8998s, CDH17-negative small cell lung cancer cell lines H524 and CORL279, and CDH17-negative human melanoma cell line M202. The chimeric monoclonal CDH17 antibody comprises the mouse anti-CDH17 antibody VH and VL regions fused to the human IgG1 constant region and the human kappa light chain constant region, respectively, where different numerical designations reflect different mouse anti-CDH17 antibody isolates. Briefly, xenograft models of different human cancer cell lines were established in six-week-old CD-1 athymic nude mice (Charles River Laboratories). When the tumors reached 150 to 300 mm 3 The mice were randomized into treatment groups after the average size of the humanized antibodies was measured. The humanized antibodies were diluted to a working concentration of 1 mg / ml in sterile saline for intravenous tail vein (IV) injection. Tumor xenografts were measured three times per week with a caliper and tumor volume (mm) was determined by multiplying height × width × length. 3 Mice were treated for 2-7 weeks. At the end of the study, animals were euthanized and tumor tissues were excised and stored separately as snap-frozen or formalin-fixed paraffin-embedded (FFPE) tissues for biomarker analysis.

[0456] The results of the xenograft assays are shown in Figures 3-10.

[0457] Figure 3 shows the results of a xenograft assay of parental mouse monoclonal antibodies against CDH17, where different anti-CDH17 antibodies had different efficacies in inhibiting the growth of CDH17-positive colorectal tumor cells SNU-C1, with little effect on the body weight of the treated mice. Figure 3A Shown are SNU-C1 (CDH17+) cell line xenografts treated with a non-targeting IgG control antibody or 6 different mAbs against CDH17. All antibodies were treated once a week by IV tail vein injection at a dose of 10 mg antibody / kg, 6 repeated doses, and 6 mice per arm. Lines represent mean tumor volume ± SEM. Figure 3B is a bar graph showing the mean change (+SEM) in tumor volume in each treatment group over 32 days of treatment, and Figure 3C The mean percent change in mouse body weight during the treatment window is shown.

[0458] FIG4 shows the efficacy of a panel of humanized CDH17 mAbs in CDH17-positive SNUC1 human CRC cell line xenografts. Figure 4A Shown are tumor volumes (mm) of SNUC1 (CDH17+) cell line xenografts treated with a non-targeting IgG control antibody or 10 different antibodies against CDH17, eight humanized anti-CDH17 antibodies (CDH17-646-h7, CDH17-653-h42, CDH17-653-h43, CDH17-657-h16, CDH17-663-h7, CDH17-670-h12, CDH17-675-h11, or CDH17-683-h6), and two parental chimeric monoclonal anti-CDH17 antibodies (CDH17-653-m and CDH17-657-m). 3 ). Mice were treated once weekly by IV tail vein injection of each antibody at a dose of 10 mg antibody / kg with 5 repeated doses. 8 mice per arm. Lines represent mean tumor volume ± SEM. Figure 4B is a bar graph showing the mean change (+SEM) in tumor volume for each treatment group over 28 days of treatment. Figure 8C Shown are the percentage changes in body weight over time following administration of a control antibody or 10 different antibodies against CDH17, eight humanized anti-CDH17 antibodies (CDH17-646-h7, CDH17-653-h42, CDH17-653-h43, CDH17-657-h16, CDH17-663-h7, CDH17-670-h12, CDH17-675-h11, or CDH17-683-h6), and two parental chimeric monoclonal anti-CDH17 antibodies (CDH17-653-m and CDH17-657-m).

[0459] Figure 5 shows the efficacy of a panel of humanized CDH17 mAbs in CDH17-positive HPAF-2 human pancreatic cell line xenografts. Figure 5A HPAF-2 (CDH17+) cell line xenografts treated with a non-targeting IgG control antibody or 8 different humanized antibodies against CDH17 are shown. All mice were treated once a week by IV tail vein injection of each antibody at a dose of 10 mg antibody / kg, with 5 repeated doses. 8 mice per arm. Lines represent mean tumor volume ± SEM. Figure 5B is a bar graph showing the mean change (+SEM) in tumor volume for each treatment group over 25 days of treatment.

[0460] Figure 6 shows that humanized CDH17 mAb has no anti-tumor activity in CDH17-negative M202 human melanoma cell line xenografts. Fig. 6A Shown are M202 (CDH17-) cell line xenografts treated with a non-targeting IgG control antibody or 5 different humanized antibodies against CDH17. All mice were treated once a week by IV tail vein injection of each antibody at a dose of 10 mg / kg, with 4 repeated doses. 8 mice per arm. Lines represent mean tumor volume ± SEM. Figure 6B is a bar graph showing the mean change (+SEM) in tumor volume for each treatment group over 24 days of treatment.

[0461] FIG. 7 shows the efficacy of the cytotoxic drug conjugate of the chimeric monoclonal antibody CDH17-ADC against the CDH17 protein in selectively inhibiting and killing a variety of different CDH17-positive human cancer cell line xenografts, while having significantly lower efficacy against CDH17-negative human cancer cell line xenografts. Fig. 7A -C shows the results of SNU-C1, PaTu8998s and LS514 (all CDH17+) cell line xenografts treated with non-targeting IgG control antibody, CDH17-653 (chimeric mAb) or CDH17-ADC generated by CDH17-653-chimeric mAb. Mice were treated with 10 mg / kg of control IgG antibody and mAb and 5 mg / kg of ADC by IV tail vein injection, with repeated doses 3 times per week. Fig.7D -F shows H524, CORL279 and M202 (all CDH17-) cell line xenografts treated with control or CDH17-ADC as described above. 6-8 mice per arm. Lines represent mean tumor volume ± SEM.

[0462] FIG8 shows the efficacy of humanized CDH17-ADC in CDH17-positive SNUC1 human CRC cell line xenografts. Fig. 8A Shown are SNUC 1 (CDH17+) cell line xenografts treated with a non-targeting IgG control antibody or 3 different CDH17-ADCs generated by humanized CDH17-mAbs (CDH17-ADC-646-h7, CDH17-ADC-653-h43, and CDH17-ADC-663-h7). Mice were administered 5 mg / kg of control antibodies and ADCs once a week by IV tail vein injection for 3 repeated doses. 8 mice per arm. Lines represent mean tumor volume ± SEM. Figure 8B is a bar graph representing the mean change (+SEM) in tumor volume for each treatment group over the 42-day study (last day of the control arm), demonstrating the effectiveness of A DCs in promoting regression of CDH17-positive human colorectal tumors in vivo. Figure 8C Shown are the percentage changes in body weight over time following administration of a control antibody or three different CDH17-ADCs generated from humanized CDH17-mAbs (CDH17-ADC-646-h7, CDH17-ADC-653-h43, and CDH17-ADC-663-h7).

[0463] FIG. 9 shows the efficacy of humanized CDH17-ADC in CDH17-positive HPAF-II human pancreatic cancer cell line xenografts. Fig.9A HPAF-II (CDH17+) cell line xenografts treated with a non-targeting IgG control antibody or 3 different CDH17-ADCs (CDH17-ADC-646-h7, CDH17-ADC-653-h43, and CDH17-ADC-663-h7) generated by humanized CDH17-mAb are shown. Mice were treated with 5 mg / kg of control antibody and ADCs by IV tail vein injection once a week for 3 repeated doses. 8 mice per arm. Lines represent mean tumor volume ± SEM. Fig. 9B is a bar graph showing the mean change (+SEM) in tumor volume for each treatment group over the 27-day study (last day in the control arm).

[0464] FIG. 10 shows that humanized CDH17-ADC has no anti-tumor activity in CDH17-negative M202 human melanoma cell line xenografts. Fig. 10AShown are M202 (CDH17-) cell line xenografts treated with a non-targeting IgG control antibody or 3 different CDH17-ADCs (CDH17-ADC-646-h7, CDH17-ADC-653-h43, and CDH17-ADC-663-h7) produced by humanized CDH17-mAb. Mice were treated with 5 mg / kg of control antibody and ADCs by IV tail vein injection once a week for 3 repeated doses. 8 mice per arm. Lines represent mean tumor volume ± SEM. Fig. 10B is a bar graph showing the mean change (+SEM) in tumor volume for each treatment group over the 31 days of the study (last day in the control arm).

[0465] The CDH17 antibody drug conjugate (ADC) provided herein has been conjugated to the cytotoxic agent MMAE via a cleavable linker VC-PAB (thus comprising VC-PAB-MMAE). The average number of MMAE per antibody is 4 as measured by HIC and / or MS. The ADC is a heterogeneous conjugate in which about 90% of the MMAE is conjugated to the interchain disulfide bond between the IgG heavy chain and the light chain (reacting with the thiol of the cysteine ​​obtained by the reduction of the interchain disulfide bond). The ADC has been prepared at Wuxi Bio using its DAR4 technology.

[0466] Fig.11 and Fig.12 The time course of internalization of unconjugated "naked" antibodies to humanized anti-CDH17 antibodies (07-0646-h7, 07-0653-h33 and / or 07-0663-h7) in CDH17-positive human cancer cell lines HPAF-II human pancreatic cancer cell line and LS513 human colorectal cancer cell line is shown in FIG. Figure 13-14 The time course of internalization of MMAE-conjugated antibodies (07-0646-h7-MMAE, 07-0653-h43-MMAE, and 07-0663-h7-MMAE) in the LS513 human colorectal cancer cell line is shown. In addition, Fig.14 Also shown is the time course of internalization of Alexa Fluor 647, AF647, conjugated 07-0663-h7 antibody (07-0663-h7-AF647) in the LS513 cell line in vitro.

[0467] All three naked antibodies and their ADCs were internalized. The internalization process could be captured by imaging 2 hours after antibody binding and was complete within 24 hours.

[0468] To determine the relative antigen binding strength of the humanized antibodies, FACS assays were performed essentially as described in Example 1. Briefly, cells overexpressing CDH17 (HEK293T cells transfected with expression plasmids for human, monkey, mouse, or rat CDH17 fused to native fluorescent protein (mGFP or mox GFP)) or untransfected HEK293T control cells were incubated at approximately 150,000 cells in 50 μL of 2% FBS / PBS with 1 μg of humanized anti-CDH17 antibodies (07-0646-h7, 07-0653-h43, and 07-0663-h7) or Alexa Fluor conjugated antibodies. The cells were incubated with an anti-human IgG Fc secondary antibody (from BioLegend, 409320) on ice for 30 min and washed with 2% FBS / PBS. The 647 anti-IgG Fc secondary antibody was incubated on ice for 30 minutes. TM C6 flow cytometer (San Jose, CA), based on the Alexa Fluor 4-H channel Antibody binding was measured by 647 fluorescence, and fusion protein overexpression was measured based on GFP fluorescence in the FL1-H channel. Fig.15 All three humanized antibodies were shown to preferentially bind to human CDH17 protein, and less to monkey (rhesus monkey) CDH17 protein. For two humanized anti-CDH17 antibodies, 07-0646-h7 and 07-0663-h7, there appeared to be little or no binding to mouse or rat CDH17 protein; whereas humanized anti-CDH17 antibody 07-0653-h3 had detectable affinity for mouse CDH17 protein and significant binding (cross-reactivity) to rat CDH17, with the binding level reaching a level similar to that of human CDH17 protein. Figure 2 shows the similarity of the amino acid sequences of CDH17 proteins from four different species.

[0469] Fig.16 The dissociation constant (K) of humanized anti-CDH17 antibody-CDH17 protein is shown. D) values, the proteins were from four different species. Cell-based antibody affinity (KD) of anti-CDH17 antibodies was measured by KinExA 4000 (Sapidyne Instrument, Boise, Idaho). Briefly, HEK293T cells overexpressing CDH17-fluorescent protein fusions (i.e., human CDH17-mGFP, monkey CDH17-mGFP, mouse CDH17-mGFP, or rat CDH17-mGFP) were detached with Versene and equilibrated overnight at 4°C with 2% FBS / DMEM containing 50 pM, 200 pM, 20 nM, or 50 nM antibodies. Cell concentrations ranged from 5×10 6 cells / mL or 1×10 7 The cells were centrifuged at 1500 rpm for 10 minutes and the supernatant was saved. PMMA beads (Sapidye Instrument, catalog number 440176) were pre-coated with 30 μg / ml goat anti-human IgG (Jackson ImmunoResearch Labs (catalog number 109-005-003). 0.5 μg / ml fluorescent secondary antibody Alexa Fluor 647 AffiniPure goat anti-human IgG (Jackson ImmunoResearchLabs, catalog number 109-605-088) was diluted in 1% BSA / PBS. Antibody solution only (signal 100%) and non-specific binding (NSB, buffer only) controls were also included in the measurement. K was calculated using the two antibody curves analyzed by n curve analysis. D .like Fig.16 As shown in Figure 2, the K values ​​of humanized anti-CDH17 antibodies 07-0646-h7 and 07-0663-h7 against human and monkey CDH17 proteins were D The K of humanized anti-CDH17 antibody 07-0653-h43 was less than about 1 nM and had no measurable binding to mouse or rat CDH17 protein. In contrast, although the humanized anti-CDH17 antibody 07-0653-h43 was unable to bind to mouse CDH17 protein, it bound to rat CDH17 protein as well as human and monkey CDH17 proteins, making its K of human, monkey, and rat CDH17 proteins D Less than about 1 nM, and the 95% confidence interval values ​​overlap.

[0470] Fig.17 The biochemical, biophysical, and cell biological characteristics of three lead humanized anti-CDH17 antibodies are summarized, along with the properties of several commercial antibodies measured under the same conditions. Fig.18The three lead anti-CDH17 antibodies (07-0646-h7, 07-0653-h43 and 07-0663-h7) were shown to maintain overall stability for a long time of 5 weeks over a wide temperature range of -80°C to 37°C, as analyzed by non-reducing SDS-PAGE of stored samples under reducing or non-reducing conditions immediately before electrophoresis.

[0471] Fig.19 The results of size exclusion chromatography and SDS-PAGE analysis of untreated or peptide-N-glycosidase F (PNGase F) treated humanized anti-CDH7 antibody 07-0646-h7 are shown. Note that the antibody preparation is fairly homogeneous with approximately 95% being monomeric and a small fraction being aggregates, which is also summarized in Fig.17 middle.

[0472] The humanized anti-CDH17 antibodies of the present invention target human CDH17 protein with nanomolar affinity and can inhibit the growth of CDH17-positive human tumors. The antibodies stain CDH17-positive cells and are internalized within a few hours, ranging from about 2 to 24 hours depending on the tumor cell line. Different human tumors have different levels of CDH17 expression ( Figure 1 ), which may affect the effectiveness of anti-CDH17 antibodies in terms of growth inhibition and antibody internalization. It has been shown that as an anti-CDH17 antibody-drug conjugate, ADC can not only inhibit the growth of CDH17-positive tumors, but also cause tumor regression, an effect not seen in CDH17-negative tumors.

[0473] Example 3

[0474] This example describes the antibody binding epitope mapping of CDH17 hAb 0663-h7.

[0475] A 15-amino acid peptide was designed that spans the ECD of CDH17 (SEQ ID NO: 51). Each peptide has a 4-amino acid offset, i.e., each peptide overlaps with the previous and / or next peptide by 11 amino acids. A total of 189 peptides were designed and manufactured by JPT Peptide Technologies using biotin labeling.

[0476] The biotinylated peptides were placed in each well of a 96-well plate and dissolved in DMSO to a concentration of about 0.45 μg / μl (each peptide had about 25 nmol per well, but the concentration would vary due to the different molecular weights of each peptide).

[0477] For ELISA, 1 μl of each biotinylated peptide plus 99 μl of peptide coating buffer (PBS containing 40% DMSO and 0.05% Tween20) was added to the wells of a microtiter plate coated with streptavidin. The culture plate was incubated overnight at 4°C without shaking. Then, the plate was washed 4 times with 300 μl of wash buffer (PBS containing 0.05% Tween20). 200 μl of blocking buffer (PBS containing 400 μM biotin and 20% sucrose) was added to each well, and the plate was incubated at room temperature for 30 minutes without shaking. After washing 3 times with wash buffer, 300 μl of SuperBlock T20 (TBS) blocking buffer (Thermo Scientific, #37536) was applied to each well and incubated for 1 hour at room temperature. After washing 3 times, a dose of 200 ng / ml of CDH17 hAb for each peptide was added to the well. Peptides showing positive signals were titrated in duplicate ELISA using CDH17 hAb starting at 500 ng / ml and going through 11 point dilutions 1 in 2 (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.906, 1.953, 0.977, 0.488 ng / ml respectively).

[0478] Titration ELISA identified overlapping peptides 18-20 with a core region of 7 amino acids (LDANGII, SEQ ID NO:53) for CDH17 hAb 07-0663-h7 binding ( Fig. 20 ).

[0479] The 7 amino acid epitope core bound by CDH17 hAb 07-0663-h7 was further analyzed to identify the amino acids that are critical for binding. Fig. 20 As shown, single point mutations were designed in the epitope of 7-mer amino acids. Biotinylated peptides were made and assayed by ELISA as described above. The mutation of 5 amino acids (DANGI, SEQ ID NO: 54) reduced or eliminated the binding activity to CDH17 hAb 07-0663-h7. Therefore, the critical minimal epitope region for antibody binding is the region of 5 amino acids DANGI ( Fig.21 ).

[0480] Example 4

[0481] Selected CDH17 antibodies 07-646-h7 and 07-663-h7 were evaluated for their ability to induce antibody-dependent cellular cytotoxicity (ADCC) in CDH17-positive cell lines.

[0482] CDH17 positive cell lines (LS513 and SNUC1) and CDH17 negative cell lines (M202) were incubated with different concentrations of CDH17 antibody or control hIgG1 from about 0.1 pM to 10 μM for 16 hours. TM NFAT-CD16 effector cells (InvivoGen, San Diego, USA) were incubated with target cells for 6 h.

[0483] As shown in Figure 22, NFAT activation was assessed by determining Lucia luciferase activity in the supernatant, which indicated the induction of ADCC response. CDH17 antibodies, particularly 07-646-h7 and 07-663-h7, induced ADCC in CDH17 positive cell lines.

[0484] Example 5

[0485] The activity of the CDH17-CD3 bispecific antibody was evaluated by a T cell activation assay using Jurkat cells carrying the NFAT-RE reporter gene.

[0486] T cell activation was measured using Promega's T cell activation bioassay kit (NFAT-RE J1621). A pair of CDH17-positive cell lines (HPAF2 and SNUC1) were seeded in each well of a white 96-well plate at a density of 20,000-40,000 cells / well and incubated overnight at 37°C, after which the thawed Jurkat T cells included in the assay kit were added to the seeded cells (the cell ratio was 1:1) and treated with CDH17-CD3 bispecific antibodies (07-0653-h43Bs, 07-0646-h7Bs, and 07-0663-h7Bs, each containing a C-terminal (HIS) 6 tag). For comparison, a control without target-positive cells was included. The treated plate was incubated at 37°C for 6 hours, then the Bio-Glo reagent (included in the kit) was added and immediately read on a Varioskan LUX plate reader. The unit is RLU.

[0487] like Fig.23 As shown, treatment of CDH17 positive cell lines (HPAF2 and SNUC1) with 07-0653-h43Bs bispecific antibodies caused T cell activation compared to no target cell controls. In contrast, for 07-0646-h7Bs and 07-0663-h7Bs bispecific antibodies, there was no difference in T cell activation levels between CDH17 positive cells and no target cell controls. The bispecific antibodies used herein also contain a C-terminal (HIS) 6 tag.

[0488] Example 6

[0489] Determined by indirect flow cytometry (Sartorius, Gottingen, Germany) evaluated the binding of bispecific antibodies and scFv fragments to CDH17-positive cells. CDH17-positive LS513 cells or CDH17-negative M202 cells were assayed using primary antibodies and fluorescent dye-labeled anti-His secondary antibodies, where the expression of FL4-H value obtained by the software.

[0490] Table 8B shows CDH17 scFvs and BiTEs that were demonstrated to selectively bind to CDH17-positive LS513 cells, but not to CDH17-negative M202 cells.

[0491] Table 1

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509] * SEQ ID NO: 86-89 (Group 1) and SEQ ID NO: 90-93 (Group 2) have both been used to express HC and LC of 07-0663-h7. The sequences in Group 1 and Group 2 differ in the use of different signal peptides (cleaved from the mature protein) and the presence of different silent nucleic acid mutations that do not change the amino acid sequence of the translated protein; SEQ ID NO 86-89 have been codon optimized.

[0510] *The scFv and bispecific antibodies (eg, BiTEs) used herein also contain a C-terminal (His)6 epitope tag, which facilitates purification and detection.

[0511] *It is well understood in the art that newly synthesized polypeptides intended for use in the secretory pathway (e.g., secreted or membrane-inserted proteins) include a signal peptide. Therefore, any of the polypeptides presented herein may also include a signal peptide, the variations of which are well known in the art.

[0512] Table 2A Amino acid sequence of humanized CDH17 antibody 07-0646-h7 (IMGT numbering)

[0513]

[0514]

[0515] Table 2B Amino acid sequence of humanized CDH17 antibody 07-0646-h7 (Kabat numbering)

[0516]

[0517]

[0518] Table 2C Amino acid sequence of humanized CDH17 antibody 07-0646-h7 (AbM numbering)

[0519]

[0520]

[0521] Table 3A Amino acid sequence of humanized CDH17 antibody 07-0653-h43 (IMGT numbering)

[0522]

[0523] Table 3B Amino acid sequence of humanized CDH17 antibody 07-0653-h43 (Kabat numbering)

[0524]

[0525]

[0526] Table 3C Amino acid sequence of humanized CDH17 antibody 07-0653-h43 (AbM numbering)

[0527]

[0528]

[0529] Table 4A Amino acid sequence of humanized CDH17 antibody 07-0663-h7 (IMGT numbering)

[0530]

[0531] Table 4B Amino acid sequence of humanized CDH17 antibody 07-0663-h7 (Kabat numbering)

[0532]

[0533]

[0534] Table 4C Amino acid sequence of humanized CDH17 antibody 07-0663-h7 (AbM numbering)

[0535]

[0536]

[0537] Table 5 Nucleic acid sequences of humanized CDH17 antibodies

[0538]

[0539]

[0540]

[0541] Table 6 Binding activity of antibodies and ADC

[0542]

[0543]

[0544] Table 7 Antibody characteristics

[0545]

[0546]

[0547] Table 8A Binding activity of antibodies determined by flow cytometry

[0548]

[0549] Table 8B CDH17 sc-fv and BiTE selectively bind to CDH17 positive cells

[0550]

[0551] Table 9 Alternative CDH17 antibody names and expression vectors used to produce CDH17 antibodies

[0552]

[0553]

[0554]

[0555] *Chimeric anti-CDH17 antibodies comprise mouse light and heavy chain variable regions and human IgG1 constant regions, wherein the human IgG1 light and heavy chain variable regions are replaced by the variable regions of mouse anti-CDH17 antibodies.

[0556] *Plasmid p07-0663-h7 containing DNA encoding humanized anti-CDH17 antibody was deposited with the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Virginia 20110-2209) on December 7, 2022, and assigned accession number _____. The deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0557] Table 10: CDH17 antibody conjugates

[0558]

[0559] @ MMAE conjugation was performed on the free thiol group of cysteine ​​residues using MC-VC-PAB-MMAE.

[0560] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0561] In the context of describing the present disclosure (especially in the context of the appended claims), the use of the terms "a", "an", "the", and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted.

[0562] Unless otherwise indicated herein, the enumeration of ranges of values ​​herein is intended only to be used as a shorthand method of individually representing each individual value belonging to the range, and each individual value and endpoint is incorporated into the specification as if individually stated herein. As used herein, when used before a numerical designation, such as temperature, time, amount, concentration, etc. (including ranges), the term "about" represents an approximate value, which can vary (+) or (-) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0563] Unless otherwise noted herein or otherwise contextually clearly contradictory, all methods described herein may be performed in any suitable order. The use of any and all embodiments or various languages ​​(e.g., "such as") provided herein is intended only to better illustrate the present disclosure, and unless otherwise required, will not limit the scope of the present disclosure. The language in this specification should not be interpreted as indicating any non-required element as essential to practicing the present disclosure.

[0564] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventor for carrying out the present disclosure. Variants of those preferred embodiments may become apparent to those of ordinary skill in the art after reading the above description. The inventor of the present case hopes that the technician will adopt these variations as appropriate, and the inventor of the present case intends to practice the present disclosure in a manner different from that specifically described herein. Therefore, as long as applicable law permits, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims attached hereto. In addition, unless otherwise indicated herein or otherwise clearly contradictory in context, the present disclosure encompasses any combination of the above-mentioned elements in all possible variations thereof.

Claims

1. An antigen binding protein, comprising: a. CDRs 1-3 derived from a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSS (SEQ ID NO: 49) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity; and / or b. CDRs 1-3 derived from a light chain variable region comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIK (SEQ ID NO: 50) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity.

2. An antigen-binding protein, comprising: a. CDRs 1-3 derived from a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTSYNMHWVRQAPGQGLEWIGAIYPGNGDTSYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGRGRYFEYWGQGTTLTVSS (SEQ ID NO: 45) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity; and / or b. CDRs 1-3 derived from a light chain variable region comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDYTLTISSVQPEDFATYYCQQYDSSPSTFGQGTKLEIK (SEQ ID NO: 46) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity.

3. An antigen binding protein, comprising: a. CDRs 1-3 derived from a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGVIYPYSGGIGYAQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGRGDYFGLFDFWGQGTTVTVSS (SEQ ID NO: 47) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity; and / or b. CDRs 1-3 derived from a light chain variable region comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRATSSLSYIHWYQQKPGKAPKPLIYEISKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWNYPFTFGQGTKLEIK (SEQ ID NO: 48) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85% sequence identity.

4. An antigen binding protein, comprising: a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFTDHT (SEQ ID NO: 13) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; b. a heavy chain CDR2 comprising the following amino acid sequence: IFPRDDIV (SEQ ID NO: 14) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; c. a heavy chain CDR3 comprising the following amino acid sequence: ARPPYYYSRNFYFDY (SEQ ID NO: 15) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; d. a light chain CDR1 comprising the following amino acid sequence: SIISSSK (SEQ ID NO: 16) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; e. a light chain CDR2 comprising the following amino acid sequence: GTS (SEQ ID NO: 17) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; f. a light chain CDR3 comprising the following amino acid sequence: QQWSNYPFT (SEQ ID NO: 18) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or g. A combination of any two or more of (a)-(f).

5. An antigen binding protein, comprising: a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFTSYN (SEQ ID NO: 1) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; b. a heavy chain CDR2 comprising the following amino acid sequence: IYPGNGDT (SEQ ID NO: 2) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGRYFEY (SEQ ID NO: 3) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; d. a light chain CDR1 comprising the following amino acid sequence: SSVSSSY (SEQ ID NO: 4) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; e. a light chain CDR2 comprising the following amino acid sequence: STS (SEQ ID NO: 5) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; f. a light chain CDR3 comprising the following amino acid sequence: QQYDSSPST (SEQ ID NO: 6) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or g. A combination of any two or more of (a)-(f).

6. An antigen binding protein, comprising: a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFTDYY (SEQ ID NO: 7) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; b. a heavy chain CDR2 comprising the following amino acid sequence: IYPYSGGI (SEQ ID NO: 8) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGDYFGLFDF (SEQ ID NO: 9) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; d. a light chain CDR1 comprising the following amino acid sequence: SSLSY (SEQ ID NO: 10) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; e. a light chain CDR2 comprising the following amino acid sequence: EIS (SEQ ID NO: 11) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; f. a light chain CDR3 comprising the following amino acid sequence: QQWNYPFT (SEQ ID NO: 12) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or g. A combination of any two or more of (a)-(f).

7. The antigen binding protein of claim 4, further comprising: a. a heavy chain FR1 comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKISCKVS (SEQ ID NO: 37) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; b. a heavy chain FR2 comprising the following amino acid sequence: IHWMRQAPGQGLEWIGY (SEQ ID NO: 38) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity; c. a heavy chain FR3 comprising the following amino acid sequence: VYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 39) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity; d. a heavy chain FR4 comprising the following amino acid sequence: WGQGTTLTVSS (SEQ ID NO: 40) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; e. a light chain FR1 comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRVS (SEQ ID NO: 41) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the following amino acid sequence: LHWYQQKPGKAPKPLIY (SEQ ID NO: 42) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; g. a light chain FR3 comprising the following amino acid sequence: TLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC (SEQ ID NO: 43) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the following amino acid sequence: FGQGTKLEIK (SEQ ID NO: 44) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or i. A combination of any two or more of (a)-(h).

8. The antigen binding protein of claim 5, further comprising: a. a heavy chain FR1 comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKAS (SEQ ID NO: 21) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; b. a heavy chain FR2 comprising the following amino acid sequence: MHWVRQAPGQGLEWIGA (SEQ ID NO: 22) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; c. a heavy chain FR3 comprising the following amino acid sequence: SYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 23) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; d. a heavy chain FR4 comprising the following amino acid sequence: WGQGTTLTVSS (SEQ ID NO: 24) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; e. a light chain FR1 comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVTMTCRAS (SEQ ID NO: 25) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the following amino acid sequence: LHWYQQKPGKAPKLLIY (SEQ ID NO: 26) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; g. a light chain FR3 comprising the following amino acid sequence: NLASGVPSRFSGSGSGTDYTLTISSVQPEDFATYYC (SEQ ID NO: 27) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the following amino acid sequence: FGQGTKLEIK (SEQ ID NO: 28) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or i. A combination of any two or more of (a)-(h).

9. The antigen binding protein of claim 6, further comprising: a. a heavy chain FR1 comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 29) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; b. a heavy chain FR2 comprising the following amino acid sequence: MNWVRQAPGQGLEWMGV (SEQ ID NO: 30) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity; c. a heavy chain FR3 comprising the following amino acid sequence: GYAQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 31) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; d. a heavy chain FR4 comprising the following amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 32) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; e. a light chain FR1 comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRAT (SEQ ID NO: 33) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the following amino acid sequence: IHWYQQKPGKAPKPLIY (SEQ ID NO: 34) or a variant sequence thereof having only one or two amino acids different or having at least or about 85%, 90% or 95% sequence identity; g. a light chain FR3 comprising the following amino acid sequence: KLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC (SEQ ID NO: 35) or a variant sequence thereof having only one or two amino acids that differ or have at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the following amino acid sequence: FGQGTKLEIK (SEQ ID NO: 36) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or i. A combination of any two or more of (a)-(h).

10. An antigen binding protein, comprising: a. a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSS (SEQ ID NO: 49) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIK (SEQ ID NO: 50) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

11. An antigen binding protein, comprising: a. a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTSYNMHWVRQAPGQGLEWIGAIYPGNGDTSYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGRGRYFEYWGQGTTLTVSS (SEQ ID NO: 45) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDYTLTISSVQPEDFATYYCQQYDSSPSTFGQGTKLEIK (SEQ ID NO:46) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

12. An antigen binding protein, comprising: a. a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGVIYPYSGGIGYAQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGRGDYFGLFDFWGQGTTVTVSS (SEQ ID NO: 47) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRATSSLSYIHWYQQKPGKAPKPLIYEISKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWNYPFTFGQGTKLEIK (SEQ ID NO:48) or a variant sequence thereof that differs in only 1-5 amino acids or has at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

13. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising: a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFXDXT (SEQ ID NO: 55), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F; b. a heavy chain CDR2 comprising the following amino acid sequence: IFPRDDIV (SEQ ID NO: 14) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; c. a heavy chain CDR3 comprising the following amino acid sequence: ARPPYYYSRNFYFDY (SEQ ID NO: 15) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; d. a light chain CDR1 comprising the following amino acid sequence: SIISSSK (SEQ ID NO: 16) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; e. a light chain CDR2 comprising the following amino acid sequence: GTS (SEQ ID NO: 17) or a variant sequence thereof that differs in only one or two amino acids; f. a light chain CDR3 comprising the following amino acid sequence: QQWSNYPFT (SEQ ID NO: 18) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or g. A combination of any two or more of (a)-(f).

14. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising: a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFXSXN (SEQ ID NO: 56), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F; b. a heavy chain CDR2 comprising the following amino acid sequence: IYPGNGDT (SEQ ID NO: 2) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGRYFEY (SEQ ID NO: 3) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; d. a light chain CDR1 comprising the following amino acid sequence: SSVSSSY (SEQ ID NO: 4) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; e. a light chain CDR2 comprising the following amino acid sequence: STS (SEQ ID NO: 5) or a variant sequence thereof that differs in only one or two amino acids; f. a light chain CDR3 comprising the following amino acid sequence: QQYDSSPST (SEQ ID NO: 6) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or g. A combination of any two or more of (a)-(f).

15. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising: a. a heavy chain CDR1 comprising the following amino acid sequence: GYTFXDXY (SEQ ID NO: 57), wherein X at position 5 is N, S, R, Q, A or T, and X at position 7 is H, W, Y or F; b. a heavy chain CDR2 comprising the following amino acid sequence: IYPYSGGI (SEQ ID NO: 8) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; c. a heavy chain CDR3 comprising the following amino acid sequence: ARGRGDYFGLFDF (SEQ ID NO: 9) or a variant sequence thereof that differs in only one or two amino acids or has at least or about 70% sequence identity; d. a light chain CDR1 comprising the following amino acid sequence: SSLSY (SEQ ID NO: 10) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; e. a light chain CDR2 comprising the following amino acid sequence: EIS (SEQ ID NO: 11) or a variant sequence thereof that differs in only one or two amino acids; f. a light chain CDR3 comprising the following amino acid sequence: QQWNYPFT (SEQ ID NO: 12) or a variant sequence thereof having only one or two amino acids different or having at least or about 70% sequence identity; or g. A combination of any two or more of (a)-(f).

16. The antigen binding protein of any of the preceding claims, wherein the variant sequence has at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

17. An antigen binding protein that specifically binds to human cadherin-17 (CDH17), the antigen binding protein comprising: a. Antibody heavy chain, containing the following amino acid sequence: QVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPY YYSRNFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:94); and b. an antibody light chain comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95).

18. An antigen binding protein as claimed in any one of the preceding claims, wherein: a. The antigen binding protein binds to human cadherin-17 (CDH17) protein (SEQ ID NO: 19); b. The antigen binding protein binds to the extracellular domain of human CDH17 with a dissociation constant (K D ) is less than about 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM; c. The antigen binding protein preferentially binds to human CDH17 relative to mouse CDH17 (SEQ ID NO: 20); d. the antigen binding protein does not bind to mouse CDH17; or e. Combinations thereof.

19. An antigen binding protein as claimed in any one of the preceding claims, which specifically binds to human cadherin-17 (CDH17) or a polypeptide comprising the amino acid sequence of any of the following: THNLQVAALDANGIIVEGPVPIT (SEQ ID NO: 82), THNLQVAALDANGII (SEQ ID NO: 83), QVAALDANGIIVEGP (SEQ ID NO: 84), LDANGIIVEGPVPIT (SEQ ID NO: 85), LDANGII (SEQ ID NO: 53) and DANGI (SEQ ID NO: 54).

20. The antigen binding protein of any one of the preceding claims, wherein binding of the antigen binding protein to CDH17 reduces, interferes with or inhibits CDH17 activity.

21. The antigen binding protein of any one of the preceding claims, which is an antibody or an antibody fragment that binds an antigen.

22. The antigen binding protein of claim 21, wherein the antibody is a monoclonal antibody.

23. The antigen binding protein of claim 21 or 22, wherein the antibody is a chimeric antibody, a human antibody or a humanized antibody.

24. The antigen binding protein of any one of claims 21-23, wherein the antibody is IgG.

25. The antigen binding protein of claim 24, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

26. The antigen binding protein of any one of the preceding claims, wherein the antigen binding protein is a bispecific antigen binding protein or a bispecific T cell engager (BiTE).

27. The antigen binding protein of claim 26, wherein the bispecific antigen binding protein or the BiTE comprises the amino acid sequence shown below: a.SEQ ID NO:45 and SEQ ID NO:46; b. SEQ ID NO:47 and SEQ ID NO:48; or c. SEQ ID NO:49 and SEQ ID NO:50, Optionally, the bispecific antigen binding protein is selected from 07-0653-h43Bs, 07-0646-h7Bs and 07-0663-h7Bs.

28. The antigen binding protein of claim 21, wherein the antigen-binding antibody fragment is selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.

29. The antigen binding protein of claim 28, wherein the scFv is 07-0653-h43scfv, 07-0646-h7scfv or 07-0663-h7scfv.

30. The antigen binding protein of any one of the preceding claims, which inhibits tumor growth in xenograft mice injected with human cancer cells.

31. The antigen binding protein of any one of the preceding claims, comprising an Fc polypeptide comprising afucosylated glycans.

32. A conjugate comprising the antigen binding protein of any one of the preceding claims or an antigen binding protein described herein.

33. The conjugate of claim 32, comprising a detectable label, a cytotoxic agent, or a chemotherapeutic agent.

34. The conjugate of claim 33, wherein the chemotherapeutic agent is an anti-mitotic agent that inhibits cell division by blocking tubulin polymerization.

35. The conjugate of claim 34, wherein the anti-mitotic agent is auristatin.

36. The conjugate of claim 35, wherein the auristatin is MMAE.

37. The conjugate of any one of claims 33-36, wherein the agent or the label is conjugated to the antigen binding protein via a cleavable linker or a non-cleavable linker.

38. The conjugate of claim 37, wherein the cleavable linker is VC-PAB.

39. The conjugate of any one of claims 32-38, wherein the antigen binding protein is an antibody.

40. The conjugate of claim 39, wherein the antibody is a monoclonal antibody.

41. The conjugate of claim 39 or 40, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

42. The conjugate of any one of claims 39-41, wherein the antibody is an IgG antibody, optionally wherein the IgG is IgG1, IgG2, IgG3 or IgG4.

43. The conjugate of any one of claims 32-42, wherein the average number of units of the agent conjugated per antigen binding protein is in the range of 1 to 8, preferably wherein the average number of units of the agent conjugated per antigen binding protein is (a) in the range of 3-8, or (b) 4.

44. The conjugate of any one of claims 32-43, wherein the conjugate is a heteroconjugate.

45. The conjugate of any one of claims 32-43, wherein the conjugate is a homogeneous conjugate.

46. ​​The conjugate of any one of claims 32-45, wherein the agent is conjugated at a specific site of the antigen binding protein.

47. The conjugate of claim 46, wherein the specific site is an unpaired cysteine ​​residue.

48. The conjugate of any one of claims 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO:45 and SEQ ID NO:

46.

49. The conjugate of any one of claims 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO:47 and SEQ ID NO:

48.

50. The conjugate of any one of claims 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 49 and SEQ ID NO:

50.

51. The conjugate of any one of claims 32-47, wherein the conjugate comprises a polypeptide conjugated to VC-PAB-MMAE, the polypeptide comprising the amino acid sequence shown in SEQ ID NO:94 and SEQ ID NO:

95.

52. A fusion protein comprising the antigen binding protein of any one of the preceding claims.

53. A nucleic acid comprising a nucleotide sequence encoding the antigen binding protein of any one of claims 1-31, the conjugate of claims 32-51, or the fusion protein of claim 52.

54. The nucleic acid of claim 53, wherein the nucleic acid is cDNA.

55. A vector (eg, an expression vector) comprising the nucleic acid of claim 53 or 54.

56. The vector of claim 55, further comprising an internal ribosome entry site (IRES).

57. A host cell comprising the nucleic acid of claim 53 or 54 or the vector of claim 55 or 56.

58. The host cell of claim 57, wherein the host cell is a bacterial cell.

59. The host cell of claim 57, wherein the host cell is a eukaryotic cell.

60. The host cell of claim 59, wherein the eukaryotic cell is a mammalian cell.

61. The host cell of claim 60, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

62. A method for producing an antigen binding protein that binds to a Cadherin-17 (CDH17) protein, the method comprising (i) culturing the host cell of any one of claims 57-61 in a cell culture medium, and (ii) harvesting the antigen binding protein from the cell culture medium.

63. A method for producing a fusion protein comprising an antigen binding protein that binds to a Cadherin-17 (CDH17) protein, the method comprising (i) culturing the host cell of any one of claims 57-61 in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.

64. A method for producing a pharmaceutical composition, the method comprising combining: (a) an antigen binding protein as described in any one of claims 1-31, a conjugate as described in any one of claims 32-51, a fusion protein as described in claim 52, a nucleic acid as described in claim 53 or 54, a vector as described in claim 55 or 56, a host cell as described in claim 57, 59 or 60, or any combination thereof; and (b) a pharmaceutically acceptable carrier, diluent and / or excipient.

65. A pharmaceutical composition comprising (a) an antigen binding protein as described in any one of claims 1-31, a conjugate as described in any one of claims 32-51, a fusion protein as described in claim 52, a nucleic acid as described in claim 53 or 54, a vector as described in claim 55 or 56, a host cell as described in claim 57, 59 or 60, or any combination thereof; and (b) a pharmaceutically acceptable carrier, diluent and / or excipient.

66. A method of treating a subject having a cancer that expresses CDH17, the method comprising administering to the subject the pharmaceutical composition of claim 65 to treat the cancer.

67. A method of inhibiting tumor growth in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 65 to inhibit tumor growth.

68. A method of reducing tumor size in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 65 to reduce tumor size.

69. A method of preventing cancer recurrence in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 65 to prevent the cancer recurrence.

70. A method of treating cancer in a subject diagnosed as a CDH17 overexpresser, the method comprising administering the pharmaceutical composition of claim 65 to the subject to prevent cancer recurrence.

71. The method of any one of claims 66-70, wherein the administration induces apoptosis of tumor cells.

72. The method of any one of claims 66-70, wherein the administration induces apoptosis of cells expressing CDH17.

73. A method for detecting E-cadherin-17 (CDH17) in a sample, the method comprising contacting the sample with an antigen binding protein as described in any one of claims 1-31, a conjugate as described in any one of claims 32-51, or a fusion protein as described in claim 52; and determining an immune complex comprising the antigen binding protein, the conjugate, or the fusion protein bound to CDH17.

74. A method for diagnosing a subject's Cadherin-17 (CDH17)-positive cancer, the method comprising contacting a biological sample comprising cells or tissues obtained from the subject with an antigen binding protein as described in any one of claims 1-31, a conjugate as described in any one of claims 32-51, or a fusion protein as described in claim 52; and assaying an immune complex comprising the antigen binding protein, the conjugate, or the fusion protein that binds to CDH17.

75. The method of claim 74, further comprising treating the subject diagnosed with a CDH17-positive cancer by administering to the subject the antigen binding protein of any one of claims 1-31, the conjugate of any one of claims 32-51, or the fusion protein of claim 52.

76. A method of producing a conjugate according to any one of the preceding claims, the method comprising a. culturing a host cell according to any one of claims 57 to 61 in a cell culture medium, wherein the host cell comprises a nucleic acid encoding an antigen binding protein or a fusion protein thereof, b. harvesting the antigen binding protein or the fusion protein from the cell culture medium, and c. linking the antigen binding protein or fusion protein to a heterologous moiety, thereby producing the conjugate, optionally wherein the heterologous moiety is a cytotoxic agent or a chemotherapeutic agent.

77. A method for activating T cells so that they target tumors or cancer cells expressing CDH17 in a subject, the method comprising administering to the subject a bispecific T cell engager (BiTE), the BiTE comprising a first scFv that binds CDH17 and a second scFv that binds CD3, wherein the first scFv that binds CDH17 comprises the VH region and VL region of the antigen binding protein of any of the preceding claims or the antigen binding protein described herein.

78. The method of claim 77, wherein the BiTE comprises an amino acid sequence as shown below: a.SEQ ID NO:45 and SEQ ID NO:46; b. SEQ ID NO:47 and SEQ ID NO:48; or c. SEQ ID NO:49 and SEQ ID NO:

50.

79. The method of claim 77 or 78, wherein the BiTE is selected from 07-0646-h7Bs, 07-0653-h43Bs and 07-0663-h7Bs, optionally wherein the BiTe is 07-0653-h43Bs.

80. A method of inducing an antibody-dependent cell-mediated cytotoxicity (ADCC) response against a tumor or cancer cell expressing CDH17 in a subject, the method comprising administering to the subject an antigen binding protein that binds CDH17, wherein the antigen binding protein comprises an Fc effector function; and the VH region and VL region of the antigen binding protein of any of the preceding claims or the antigen binding protein described herein.

81. The method of claim 80, wherein the antigen binding protein that binds CDH17 is 07-646-h7 or 07-663-h7.

82. The method of any of the preceding claims, wherein the subject is a mammal, optionally a dog, cat, mouse, or human.

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