Anti-CDH17 / EGFR / CD16a trispecific antibody and use thereof

Abstract

The present invention relates to a new CDH17 antibody, a bispecific antibody constructed on the basis thereof and specifically binding to CDH17 and EGFR, or a CDH17, EGFR and CD16A transpacific antibody, a pharmaceutical composition comprising same, a preparation method, and a use.

Description

Anti-cdh17 / egfr / cd16a trispecific antibodies and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410965516.3, filed on July 18, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present application relates to a new CDH17 antibody, and a bispecific antibody specifically binding to CDH17 and EGFR, or a trispecific antibody to CDH17, EGFR and CD16A constructed based thereon, as well as a pharmaceutical composition comprising the same, a preparation method and uses thereof.

[0004] BACKGROUND

[0005] CDH17, also known as Cadherin-17, is a member of the calcium adhesion protein superfamily, mainly expressed in intestinal epithelial cells, and has high expression levels in the small intestine and colon. It plays an important role in maintaining the integrity of epithelial tissue, and is closely related to the occurrence and development of various gastrointestinal tumors. The extracellular domain of CDH17 is composed of 7 repeat sequences, belonging to a subclass of 7D-cadherin family. It is different from typical Cadherin in structure, with a shorter intracellular domain that does not interact with other cytoplasmic components, but still maintains its adhesion function. The functions of CDH17 include acting as a polypeptide transporter and a cell adhesion molecule. It is mainly localized on the lateral and basolateral membranes of cells in the intestinal epithelium, and may achieve intercellular adhesion by directly connecting with cell scaffolds.

[0006] In tumor tissues, CDH17 is overexpressed in gastric cancer, pancreatic cancer, liver cancer, esophageal cancer, cholangiocarcinoma and colorectal cancer. Studies have shown that overexpression of CDH17 is associated with increased proliferation, migration and metastasis of tumor cells. Due to the high expression of CDH17 in various tumors, it has become a potential therapeutic target. Currently, drug development targeting CDH17 includes bispecific antibodies, CAR-T therapy and antibody drug conjugates (ADC), etc.

[0007] CD16A is a Fc gamma receptor mainly expressed on natural killer (NK) cells and involved in the process of antibody-dependent cell-mediated cytotoxicity (ADCC). In ADCC, CD16A activates NK cells by recognizing the Fc portion of IgG antibodies bound to target cells, which in turn release cytotoxic molecules leading to the death of target cells. This role of CD16A is very important in tumor immunotherapy, as it can enhance the efficacy of monoclonal antibody drugs.

[0008] Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein belonging to the ErbB receptor family with a molecular weight of about 180 kDa and tyrosine protein kinase activity. EGFR plays a role in various cellular physiological processes, including cell proliferation, differentiation, and survival. It is activated by binding to ligands such as epidermal growth factor (EGF) and transforming growth factor alpha (TGFa), which in turn triggers the activation of intracellular signaling pathways. Activation of EGFR involves receptor dimerization and autophosphorylation, which activates downstream signaling pathways including MAPK, Akt, and JNK, which control cell proliferation, differentiation, and survival. Abnormal expression or mutation of EGFR is closely related to the occurrence and development of various tumors, including glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer. In tumor treatment, high expression of EGFR is usually associated with poor prognosis, increased resistance to chemotherapy.

[0009] Cetuximab, also known as ERBITUX or Erbitux, is an IgG1 type human / mouse chimeric monoclonal antibody targeting EGFR. It specifically binds to the extracellular region of EGFR, competitively blocking the binding of ligands, thereby inhibiting the activation of EGFR and downstream signaling, reducing the proliferation of cancer cells and inducing apoptosis. Cetuximab can also kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and induce autophagy of tumor cells at the genetic level. Since its first launch in 2003, Cetuximab has been approved for the treatment of various tumors, including metastatic head and neck cancer, non-metastatic head and neck cancer, metastatic colorectal cancer, and non-small cell lung cancer. In addition, for patients with KRAS gene wild type colorectal cancer, Cetuximab can be used as a first-line treatment.

[0010] Despite the fact that cetuximab, as a monoclonal antibody targeting EGFR, has shown significant efficacy in the treatment of multiple tumors and has become an important component of tumor targeted therapy, due to the expression of EGFR in normal tissues (of which the normal skin tissue has a very high expression of EGFR), cetuximab has a strong skin toxicity (Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 2004 Jul 22; 351(4):337-45. doi: 10.1056 / NEJMoa033025. PMID: 15269313.). In the toxicology experiment of cetuximab, at the dose of 75 mg / kg, half of the marmoset monkeys died. In the clinical experiment, nearly 80% of the patients had skin-related toxicity. The toxicity limits the dose of EGFR monoclonal antibody, thereby limiting its efficacy. Moreover, the expression of EGFR in colorectal cancer is not high in general, and the EGFR monoclonal antibody is not good at enriching in the tumor, which limits its effect of inhibiting the EGFR signal. In addition, the wild-type IgG used by cetuximab has a weak antibody-dependent cell-mediated cytotoxicity (ADCC) effect, and the effect of killing tumors is not good.

[0011] Therefore, there is a need in the art to develop new antibodies capable of targeting EGFR with better efficacy (e.g., compared to cetuximab) while having improved toxicity compared to cetuximab. SUMMARY

[0012] The present application provides a new CDH17 antibody or antigen-binding fragment thereof, including multispecific antibodies, such as bispecific antibodies or trispecific antibodies, constructed based thereon.

[0013] The present application provides a new affinity-matured CD16A antibody or antigen-binding fragment thereof, including multispecific antibodies, such as bispecific antibodies or trispecific antibodies, constructed based thereon.

[0014] The present application provides a bispecific antibody that specifically binds to CDH17 and EGFR, such as a bispecific antibody constructed based on the anti-CDH17 antibody of the present application.

[0015] The present application also provides a trispecific antibody that specifically binds to CDH17, EGFR and CD16A, constructed based on the CDH17 antibody and the CD16A antibody of the present application.

[0016] The multi-specific antibody of the present application has the following characteristics:

[0017] (1) The pair design of EGFR and CDH17 is because CDH17 is highly expressed on some tumors such as colorectal cancer, and is not expressed on normal cells such as skin, so that the bispecific or trispecific antibody of the present application which specifically binds EGFR and CDH17 can achieve the effect of enrichment in tumor cells without killing normal cells;

[0018] (2) The anti-CDH17 arm of the bispecific or trispecific antibody of the present application which specifically binds EGFR and CDH17 can bind to CDH17 highly expressed on some tumors such as colorectal cancer cells with high affinity, which allows the anti-EGFR arm to bind to EGFR expressed on tumor cells for a long time, and inhibits its signaling pathway to achieve better tumor killing effect;

[0019] (3) The trispecific antibody of the present application which specifically binds EGFR and CDH17 and CD16A contains a high-affinity anti-CD16A nanobody (K D about 1 x 10 -9 ), which can efficiently bind to CD16A protein on the surface membrane of natural killer (NK) cells and pull NK cells to tumor cells highly expressing CDH17 and EGFR for killing.

[0020] In some embodiments, the trispecific antibody of the present application has one or more of the following superior properties:

[0021] The EGFR-mediated tumor inhibition effect of the trispecific antibody of the present application is stronger than that of cetuximab in in vitro and in vivo experiments;

[0022] The ADCC tumor killing effect of the trispecific antibody of the present application is stronger than that of cetuximab in in vitro and in vivo experiments.

[0023] The toxicity of the trispecific antibody of the present application is lower than that of cetuximab. In pre-toxicity experiments, no monkeys died at a dose of 150 mg / kg, whereas cetuximab killed half of the monkeys at half the dose (75 mg / kg). BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 shows the structure of CDH17 / EGFR / CD16A trispecific antibody;

[0025] Figure 2 shows humanized CD16A NK cell killing experiment;

[0026] Figure 3 shows the expression of CDH17 and EGFR in different CRC cell lines;

[0027] Figure 4 shows that the proliferation inhibition effect of EGFR / CDH17 vl bispecific antibody is better than that of control in wild-type KRAS and mutant KRAS CRC cell lines;

[0028] Figure 5 shows the structure of EGFR / CDH17 bispecific antibody and EGFR / CDH17 / CD16A trispecific antibody;

[0029] Figure 6 shows the in vitro killing results of EGFR / CDH17 of different structures;

[0030] Figure 7 shows that the proliferation inhibition effect of P006 is superior to that of cetuximab in wild-type KRAS colorectal cancer cell lines;

[0031] Figure 8 shows that the proliferation inhibition effect of P006 is superior to that of cetuximab in G12 mutant KRAS colorectal cancer cell lines;

[0032] Figure 9 shows that the ADCC effect of P006 is superior to that of cetuximab in wild-type KRAS colorectal cancer cell lines;

[0033] Figure 10 shows that the ADCC effect of P006 is superior to that of cetuximab in G12 mutant KRAS colorectal cancer cell lines;

[0034] Figure 11 shows that P006 antibody and KRAS-G12C small molecule inhibitor (top) or KRAS-G12D small molecule inhibitor (bottom) have synergistic antitumor effect;

[0035] Figure 12 shows the expression detection of CDH17 and EGFR in human skin squamous cell line (A431) and human skin keratinocyte (HaCat) cell line;

[0036] Figure 13 shows that the in vitro safety of P006 antibody is superior to that of cetuximab;

[0037] Figure 14 shows the efficacy experiment of P006 and cetuximab in NCI-H508 in vivo model;

[0038] Figure 15 shows that P006 and Fruquintinib show synergistic antitumor effect in KRAS-G12D mutant colorectal cancer mouse model;

[0039] Figure 16 shows the binding ability of antibody 68A7E3 and Hz68A7E3 to C99 cells. DETAILED DESCRIPTION

[0040] I. DEFINITIONS

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs.

[0042] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural; and vice versa. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0043] The term "about" or "approximately," when used in connection with a numerical value, means encompassing numerical values falling within 10% of the specified numerical value, preferably encompassing numerical values falling within 5% of the specified numerical value, and more preferably encompassing numerical values falling within 4%, 3%, 2%, or 1% of the specified numerical value.

[0044] As used herein, the term "and / or" means any one of the items, or any combination of two or more of the items.

[0045] In the present text when referring to "first" "second" only two domains or two chains are distinguished, without indicating the position of the two domains in any way.

[0046] As used herein, the term "comprising" or "including," or "having" means including, but not limited to, the elements, integers, or steps set forth in the specification. In the present text, when using the term "comprising" or "including", or "having" in the

[0047] The term "EGFR" as used herein includes, for example, a protein having an amino acid sequence as set forth in UniProtKB database or EMBL Interpro database under protein ID: P00533. In some embodiments, the EGFR is a human EGFR. Reference to EGFR herein also encompasses variant forms thereof, e.g. natural variants thereof. As used herein, an "antibody that binds EGFR" or "anti-EGFR antibody" or "antibody that specifically binds EGFR" refers to an antibody that is capable of binding EGFR with appropriate affinity. In some aspects, the anti-EGFR antibodies described herein also encompass multi-specific antibodies, e.g. bi-specific antibodies or tri-specific antibodies, that specifically bind EGFR and another target.

[0048] The term "CDH17" as used herein includes, for example, having an amino acid sequence as set forth in UniProtKB database or EMBL Interpro data Protein ID: Q12864. In some embodiments, CDH17 is human CDH17. Reference to CDH17 herein also encompasses variant forms thereof, for example natural variant forms thereof. As used herein, an "antibody that binds CDH17" or "anti-CDH17 antibody" or "an antibody that specifically binds CDH17" refers to an antibody that is capable of binding CDH17 with appropriate affinity. In some aspects, anti-CDH17 antibodies described herein also encompass multispecific antibodies, e.g., bispecific antibodies or trispecific antibodies, that simultaneously specifically bind CDH17 and another target.

[0049] The term "CD16A" as used herein includes, for example, having an amino acid sequence as set forth in UniProtKB database or EMBL Interpro database Protein ID: P08637. In some embodiments, CD16A is human CD16A. Reference to CD16A herein also encompasses variant forms thereof, for example natural variant forms thereof. As used herein, an "antibody that binds CD16A" or "anti-CD16A antibody" or "an antibody that specifically binds CD16A" refers to an antibody that is capable of binding CD16A with appropriate affinity. In some aspects, anti-CD16A antibodies described herein also encompass multispecific antibodies, e.g., bispecific antibodies or trispecific antibodies, that simultaneously specifically bind CD16A and another target.

[0050] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Thl, Th2, and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes).

[0051] General information on nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0052] The multispecific antibodies of the present application can comprise a linker. The term "linker" as used herein refers to any molecule capable of directly linking different portions of a multispecific antibody. Examples of linkers that establish covalent links between different portions of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol, polypropylene glycol. In some embodiments, the term "peptide linker" according to the present application refers to a sequence of amino acids, wherein the sequence links together the amino acid sequences of the individual portions of a multispecific antibody. Preferably, the peptide linker has a length sufficient to link two entities in such a way that they maintain their conformation relative to each other so that the desired activity is not hindered. The peptide linker can include or not include the following amino acid residues predominantly: Gly, Ser, Ala, or Thr. In some embodiments, the peptide structure is (G) n wherein n = any integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0053] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Native immunoglobulin "Fc domains" contain two or three constant domains, i.e., CH2, CH3, and optionally CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of a heavy chain derived from an IgG, IgA, and IgD class antibody; or the second, third and fourth constant domains (CH2, CH3 and CH4 domains) of a heavy chain derived from an IgM and IgE class antibody. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or the heavy chain constant region is according to the EU numbering scheme (also referred to as EU index) as set forth in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of an Fc region can or can not be present. Two Fc regions can dimerize to constitute a dimeric Fc, two different Fc heterodimerize to form a heterodimeric Fc. In this context, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and the light chain variable region VL, and the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin, but can include the hinge region N-terminal to the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Asp231 to the carboxy-terminus of the heavy chain. In this context, unless otherwise specified, an Fc region of the present application refers to an Fc region extending from Asp221 to the carboxy-terminus of the heavy chain. In one embodiment, a human IgGl Fc region polypeptide (including the hinge region) comprises the amino acid sequence set forth in SEQ ID NO: 57 or 58.

[0054] In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises all or a portion of a human constant region. Antibody Fc regions are directly involved in complement activation, Clq binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgGl subclass. In one embodiment, the Fc region is a human IgGl, IgG2, IgG3, or IgG4 Fc region.

[0055] In the present context, "heterodimeric Fc scaffold" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be linked at their N- or C-terminus to a domain that binds an antigen (e.g. a heavy and / or light chain variable region of an antibody or an antigen binding fragment of an antibody that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) for the construction of a multispecific antibody, e.g. a bispecific or trispecific antibody.

[0056] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, CH1 can comprise a portion of the hinge region. In some embodiments, the Fc region can comprise a portion of the hinge region.

[0057] In the present context, specific amino acid residues of an antibody IgG constant region are referred to according to the EU numbering system. For example, "S364" refers to the serine at EU position 364. Amino acid mutations at specific positions of the constant region are denoted by (original amino acid, amino acid position, mutated amino acid). For example, "S364R" refers to the substitution of the serine (S) at EU position 364 by an arginine (R). When referring to a combination of mutations, the combined mutations are connected by a plus sign (+) or "and" or "or". "S364R+D399K", S364R-D399K or "S364R and D399K" indicates that the Fc region comprises both the mutations S364R and D399K. When multiple mutation possibilities are possible at a specific position, this is indicated in the present context by the symbol " / ". For example, the mutation "K370T / S" indicates that the residue K at position 370 can be replaced by a T or S residue.

[0058] For polypeptide sequences, "conservatively modified variations" include substitutions, deletions or additions to a polypeptide sequence, which do not substantially alter the functional activity of the polypeptide sequence. For example, conservative substitutions commonly involve the exchange of a member of one amino acid for a chemically similar amino acid. Tables of conservative substitutions are well known in the art. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the desired antigen binding characteristics of the antibody molecules or binding protein molecules comprising the amino acid sequence of the present application. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100-110% or more, binding affinity to the antigen of interest relative to the parent antibody or binding protein.

[0059] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0060] The terms "VHH" or "VHH antibody" or "single domain antibody" or "single domain antibody" are used interchangeably herein and generally refer to an antibody comprising or consisting of only one heavy chain variable region having activity of binding to an antigen. A VHH typically comprises three CDRs and four highly conserved framework regions, and typically has the structure of the formula: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and CDR1 to CDR3 refer to complementarity determining regions 1-3. The CDR sequences in a VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section. A VHH typically includes only the heavy chain variable domain derived from a heavy chain antibody lacking light chains, also known as a nanobody. The VHH used in the present application is preferably from a camelid, such as a llama, or is a humanized version or a sequence optimized version (e.g., an affinity matured version to increase binding affinity) thereof. In some embodiments, the VHH of the present application is a monovalent monospecific polypeptide molecule consisting of or consisting essentially of a single heavy chain variable region (e.g., a heavy chain variable region of a heavy chain antibody).

[0061] The single domain antibodies or VHHs of the application can also be comprised in larger polypeptides / proteins. Examples of polypeptides / proteins comprising a VHH of the application include, but are not limited to, heavy chain antibodies (HcAbs) or multispecific antibodies or immunoconjugates.

[0062] A "heavy chain antibody" according to the application refers to an antibody without light chains, e.g. which can comprise from N- to C-terminus VH-Fc or VH-CH2-CH3 or VH-hinge-CH2-CH3, or VH-CH1-CH2-CH3. The heavy chain antibodies according to the application can also encompass homo-dimers, e.g. heavy chain dimer antibodies without light chains. The VH in the heavy chain antibody can be derived from a standard antibody or from a single domain antibody. For example, the VH in the heavy chain antibody can be a VHH. In some embodiments, the heavy chain antibodies according to the application can be heavy chain antibodies with a framework region and / or a heavy chain constant region derived from a camelid (llama, camel, in particular alpaca), a humanized version thereof or a sequence optimized version thereof (affinity matured version), or a fragment thereof (e.g. a fragment comprising at least part of the constant region). The heavy chain antibodies according to the application also encompass antibodies formed after fusion of a heavy chain variable region or VHH with a heavy chain constant region (e.g. a human IgG heavy chain constant region) or a Fc region (e.g. a Fc region of a human IgG, e.g. of human IgG1, IgG2, IgG3 or IgG4).

[0063] When referring to "VHH" in the context of a heavy chain antibody or multispecific antibody or fusion protein, it is understood that it is part of the multispecific antibody and not as a separate molecule.

[0064] The term "binding molecule" refers to any molecule capable of specific binding to a target, such as an antibody or antigen binding fragment thereof or a fusion protein.

[0065] The term "target" refers to the bound object to which a binding molecule is directed. The target can be an antigen, or a ligand or a receptor. The term "antigen" refers to a molecule that elicits an immune response. Such an immune response can involve antibody production or activation of specific immune cells, or both. The skilled person will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA. The term "epitope" as used herein refers to the part of an antigen that specifically interacts with an antibody molecule. When the binding molecule of the application involves an antibody-derived target binding region, "target" and "antigen" can be used interchangeably. In some embodiments, the antigen is CDH17. In some embodiments, the antigen is CD16A. In some embodiments, the antigen is EGFR. In some embodiments, the antigen is CDH17 and EGFR. In some embodiments, the antigen is CDH17, CD16A and EGFR.

[0066] The term "target binding region" as used herein refers to the part of a binding molecule, e.g. a multispecific binding molecule or a bispecific binding molecule, which binds to a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region can or can not have a tertiary structure independent of the remainder of the binding molecule and can or can not bind its target as a separate entity. The target binding region can also be a receptor or a ligand, or a domain of a receptor which is capable of binding a ligand. In case of a multispecific antibody or a bispecific antibody, the "target binding region" is also referred to as "antigen binding region".

[0067] The term "antigen binding region" as used herein refers to any part of an antibody or antigen binding fragment thereof, e.g. of a multispecific antibody or a bispecific antibody, which binds to a specific target or antigen. The antigen binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such an antigen binding region can or can not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody and can or can not bind its antigen / epitope as a separate entity. In case the binding molecule of the application relates to a target binding region derived from an antibody, "target binding region" and "antigen binding region" can be used interchangeably.

[0068] The term "multispecific binding molecule" refers to a multispecific binding molecule which is at least bispecific, e.g. a trispecific binding molecule, i.e. the molecule comprises at least a first target binding region and a second target binding region and a third target binding region, wherein the first target binding region binds to one target or antigen and the second target binding region binds to another antigen or target and the third target binding region binds to another antigen or target. The multispecific binding molecule according to the application also encompasses multispecific binding molecules comprising a plurality of target binding regions / binding sites. In some embodiments, the multispecific binding molecule of the application is a multispecific antibody. In some embodiments, the bispecific binding molecule of the application is a bispecific antibody. In some embodiments, the trispecific binding molecule of the application is a trispecific antibody.

[0069] The term "monospecific" as used herein refers to a polypeptide / protein molecule having one or more target binding regions, each of which binds to the same site or structure of the same target or to the same epitope of the same antigen.

[0070] As used herein, the term "multispecific" antibody refers to an antibody that has at least two antigen binding regions, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen. In one embodiment, provided herein is a trispecific antibody that has binding specificity for a first antigen and a second antigen and a third antigen.

[0071] When reference is made to a "first antigen binding region" in a multispecific antibody or bispecific antibody, it is meant to refer to a binding region that binds to a first antigen, and is not intended to limit the number of such antigen binding regions contained in the antibody, e.g., a multispecific antibody or bispecific antibody can contain one or more than one first antigen binding region. For example, a bispecific antibody contains a first antigen binding region and a second antigen binding region, but can contain one or more than one first antigen binding region and one or more than one second antigen binding region. For example, a trispecific antibody contains a first antigen binding region and a second antigen binding region and a third antigen binding region, but can contain one or more than one first antigen binding region and one or more than one second antigen binding region and one or more than one third antigen binding region.

[0072] When reference is made to a "target or antigen binding region being derived from an antibody," it is meant that the binding domain that makes up the target / antigen binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, e.g., a fragment of the antigen binding region that specifically binds to the antigen is or is derived from the corresponding fragment of the antibody, e.g., Fab, or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen binding region are CDRs of the antibody.

[0073] The term "derived from" means that a fragment in an antigen binding region is essentially identical to the fragment of the antibody from which it is derived, but has a mutation, e.g., a substitution, deletion or addition, at one or more positions. In a particular embodiment, the mutation is not in a CDR of the antibody.

[0074] The terms "whole antibody" or "full-length antibody" are used interchangeably herein to refer to an antibody molecule having a native immunoglobulin molecule structure. In the case of a conventional four-chain IgG antibody, the full-length antibody comprises two heavy chains (H) and two light chains (L) which are interconnected by disulfide bonds. In the case of a heavy chain antibody which has only heavy chains but lacks light chains, the full-length antibody comprises two heavy chains (H) which are interconnected by disulfide bonds. For a conventional four-chain IgG antibody, the full-length antibody heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains, CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region comprises one domain, CL. Each heavy chain variable region VH and each light chain variable region VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0075] The term "antigen-binding fragment" of an antibody is a molecule other than a full-length antibody that comprises a portion of a full-length antibody that is capable of binding to an antigen bound by the full-length antibody or competing with the full-length antibody (i.e. with the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. In addition, pepsin digestion of a full-length antibody under denaturing conditions generates F(ab')2, which is a dimer of two Fab' fragments linked by a disulfide bond from the hinge region. F(ab')2 can be reduced under non-denaturing conditions to break the disulfide bridge of the hinge region, thereby converting the F(ab')2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab fragment with the hinge region. Fv fragments consist of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be coded for by separate genes, but they can also be made using recombinant methods using a synthetic linker that connects the two domains to be produced as a single protein chain, and in which the VL and VH regions pair to form a single chain Fv (scFv) in the single protein chain.

[0076] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment that consists of two polypeptide chains, comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CHI, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N- to C-terminus, VH and one constant region selected from CHI and CL, and the other polypeptide chain comprises, from N- to C-terminus, VL and the other constant region selected from CL and CHI, wherein the VH domain and the VL domain pair to form an antigen binding site. In the present context, the Fab polypeptide chain comprising the heavy chain constant domain CHI is also referred to as "Fab heavy chain"; correspondingly, the Fab polypeptide chain comprising the light chain constant domain CL is also referred to as "Fab light chain".

[0077] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of a heavy chain and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment schemes, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same numbered positions, e.g., Kabat numbered positions, as a reference CDR sequence, e.g., any of the exemplary CDRs of the present application.

[0078] In some embodiments, the CDRs of the heavy chain variable region of the antibodies in the application are determined according to Kabat or Chothia, or a combination of Kabat and Chothia (hereinafter "Kabat & Chothia", e.g. HCDR1 corresponds to H26-H35 in the Kabat numbering system). In some embodiments, the CDRs of the light chain variable region of the antibodies in the application are determined according to Kabat.

[0079] In one embodiment, the HCDR1 in the anti-CDH17 antibodies of the application is defined according to the Kabat & Chothia rules, the HCDR2, HCDR3 are defined according to the Kabat rules, and the LCDRs are defined according to the Kabat rules, respectively.

[0080] The calculation of sequence identity between sequences is performed as follows.

[0081] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first and second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one preferred embodiment, for comparison purposes, the length of an aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0082] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while being less immunogenic when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterparts (i.e., replacing the portion of the variable region not involved in binding with the corresponding portion of a human antibody).

[0083] As used herein, the terms "against," "binds," or "binds specifically" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a particular target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biolayer interferometry assay or MSD assay or surface plasmon resonance (SPR) method.

[0084] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (K dis and K on , respectively). Affinity can be measured by common methods known in the art. One particular method for measuring affinity is the ForteBio kinetic binding assay herein.

[0085] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom.

[0086] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector associated with a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0087] The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0088] The term "treatment" refers to slowing, interrupting, arresting, ameliorating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indicia of a disease, or relieving the symptoms or inhibiting or arresting further development of the disease, condition, or disorder.

[0089] The term "prevention" includes inhibition of the occurrence or development of a disease or disorder, or symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0090] The term "effective amount" refers to that amount or dose of an antibody or fragment or composition or combination of the application, which elicits the intended effect in the patient to be treated or prevented, upon administration of a single or multiple doses. Depending on the intended effect, this can include both "therapeutically effective amounts" and "prophylactically effective amounts". A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or fragment or composition or combination of the application are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Since a prophylactic dose is used prior to or at an earlier stage of disease, it will generally be less than a therapeutic dose.

[0091] The term "therapeutic agent" as described herein encompasses any substance that is effective in preventing or treating a tumor, e.g., a cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies (e.g., immune checkpoint molecule antibodies), small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants or agonists). In some embodiments, the therapeutic agent is a small molecule drug.

[0092] The term "cytotoxic agent" as used herein refers to an agent that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0093] A "chemotherapeutic agent" includes a chemical compound useful in the treatment of a tumor, e.g., a cancer, or an immune system disorder.

[0094] The term "small molecule drug" refers to an organic compound of low molecular weight that is capable of modulating a biological process, in particular altering or preventing a pathological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD and preferably less than 1 kD, more preferably less than 500 D. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more able to penetrate cells, less susceptible to degradation, and less likely to elicit an immune response than large molecules.

[0095] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0096] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The cancer can be in an early, intermediate, or advanced stage or be a metastatic cancer.

[0097] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Tumor" encompasses solid tumors and blood-born tumors (including lymphoma) as well as metastatic lesions. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0098] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities, to treat a disease described herein. For example, such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration also encompasses use of each of the therapeutic agents in a sequential manner. In either case, the treatment regimen will provide beneficial effects from the drug combination in treating the disorder described herein.

[0099] The term "pharmaceutical combination" or "combination product" refers to a non-fixed combination or a fixed combination, including, but not limited to, a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present application, and (ii) the other therapeutic agent) are presented in a separate entity and are administered to a patient simultaneously, without specific time limitations, or sequentially with no specific time limits between administrations, wherein such administration provides therapeutically effective levels of the two or more active agents in the patient. The term "fixed combination" means that the two or more active agents are administered to the patient as a single entity. Preferably, the dosages of the two or more active agents and / or the time interval between the administration of the individual components are selected so that the use of the combination results in a synergistic effect that is greater than the effect of either active agent used alone. It will be appreciated that when "pharmaceutical combination" is mentioned herein, the individual components can be in one kit or composition or in separate formulations and not in a kit. The individual components can each be in the same or different formulation.

[0100] The term "pharmaceutical composition" refers to a composition that is in a form suitable for administration to a subject and that includes an active ingredient in an effective amount for achieving the intended biological activity not including additional ingredients that are unacceptable to the subject to which the composition is administered. In some embodiments, the pharmaceutical composition can be in the form of a formulation.

[0101] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active agent is administered.

[0102] "Subject / patient / individual sample" refers to a collection of cells or fluid obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, like from a fresh, frozen and / or preserved organ or tissue sample or biopsy or puncture sample; a bodily fluid, such as tear fluid, vitreous fluid, cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid.

[0103] The term "label" as used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope label or fluorescent label) or, in the case of an enzymatic label, catalyzes chemical changes to a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., bonding) of a detectable substance to the probe or antibody, as well as indirect labeling by conjugation with another reagent which itself is directly labeled or is known to associate with a detectable substance.

[0104] II. Antibody molecules

[0105] II-1 Anti-CDH17 antibodies of the invention or antigen-binding fragments thereof

[0106] In some embodiments, the anti-CDH17 antibodies of the invention or antigen-binding fragments thereof specifically bind to CDH17 (e.g., human CDH17 or cynomolgus CDH17). In some embodiments, the anti-CDH17 antibodies of the invention or antigen-binding fragments thereof have a binding affinity for CDH17 (e.g., human CDH17 or cynomolgus CDH17, particularly domain 1-2 of human CDH17, e.g., comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 52) with a K D value of less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM, or between recited values. In some embodiments, the anti-CDH17 antibodies of the invention or antigen-binding fragments thereof have a binding affinity for CDH17 (e.g., human CDH17 or cynomolgus CDH17, particularly domain 1-2 of human CDH17) with a K D value of greater than or equal to about 1 nM. In some embodiments, the antibody binding affinity is determined by a biolayer interferometry technique, e.g., Octet® (ForteBio).

[0107] In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof are capable of specifically binding to CDH17, e.g., human or cynomolgus CDH17, e.g., domains 1-2 of human CDH17.

[0108] In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof comprise 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof comprise 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof comprise 3 complementarity determining regions from a heavy chain variable region (HCDR) and 3 complementarity determining regions from a light chain variable region (LCDR).

[0109] In some aspects, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof comprise a heavy chain variable region (VH). In some aspects, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof comprise a light chain variable region (VL). In some aspects, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof comprise a heavy chain variable region and a light chain variable region (VH / VL). In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3.

[0110] In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof further comprise an antibody heavy chain constant region. In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof further comprise an antibody light chain constant region. In some embodiments, the anti-CDH17 antibodies of the application or antigen-binding fragments thereof further comprise a heavy chain constant region and a light chain constant region.

[0111] In some embodiments, the heavy chain variable region:

[0112] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 21 or 25; or

[0113] (ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or 25; or

[0114] (iii) comprises an amino acid sequence which has 1 or more, preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1, amino acid alteration(s) (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 21 or 25, preferably the amino acid alteration(s) do not occur in the CDR regions.

[0115] In some embodiments, the light chain variable region:

[0116] (i) comprises or consists of an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 16 or 26; or

[0117] (ii) comprises or consists of an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 16 or 26; or

[0118] (iii) comprises an amino acid sequence which has 1 or more, preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1, amino acid alteration(s) (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 16 or 26, preferably the amino acid alteration(s) do not occur in the CDR regions.

[0119] In some embodiments, the three complementarity determining regions from the heavy chain variable region (HCDRs), HCDR1, HCDR2 and HCDR3 are selected from

[0120] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 21 or 25;

[0121] (ii) a sequence which comprises at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration(s) (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions compared to the sequence of any of (i),

[0122] wherein the HCDR1, 2 and 3 can be determined according to any CDR-determining scheme, respectively, e.g., according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, respectively, or a combination thereof;

[0123] For example, the HCDR1 sequence is defined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 sequences are defined according to the Kabat scheme, respectively.

[0124] In some embodiments, the three complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2 and LCDR3 are selected from

[0125] (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as shown in SEQ ID NO: 16 or 26, or

[0126] (ii) a sequence comprising collectively, over the three LCDR regions, no more than 5, 4, 3, 2, or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) relative to the sequence of any of (i),

[0127] wherein the LCDR1, 2 and 3 can each be determined according to any scheme for determining CDRs, e.g., according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, respectively, or a combination thereof;

[0128] For example, the LCDR1, 2 and 3 are each determined according to the scheme of Kabat.

[0129] In some embodiments, the anti-CDH17 antibody or antigen binding fragment thereof described herein comprises:

[0130] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as shown in SEQ ID NO: 21 or 25, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as shown in SEQ ID NO: 16 or 26;

[0131] (ii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as shown in SEQ ID NO: 21, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as shown in SEQ ID NO: 16;

[0132] (iii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as shown in SEQ ID NO: 25, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as shown in SEQ ID NO: 26;

[0133] wherein the HCDR1, 2 and 3 and LCDR1, 2 and 3 can each be determined according to any scheme for determining CDRs, e.g., according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, respectively, or a combination thereof;

[0134] For example, the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are determined according to the Kabat scheme, respectively, and the LCDR1, 2 and 3 are determined according to the Kabat scheme, respectively.

[0135] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 22, or the HCDR1 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 22.

[0136] In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 23 or 27, or the HCDR2 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 23 or 27.

[0137] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 24, or the HCDR3 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 24.

[0138] In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, or the LCDR1 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 17.

[0139] In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18, or the LCDR2 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 18.

[0140] In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 19, or the LCDR3 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 19.

[0141] In some embodiments, the anti-CDH17 antibody or antigen-binding fragment thereof of the present application comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23 or 27, SEQ ID NO: 24, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, respectively.

[0142] In some embodiments, the anti-CDH17 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region VH comprises a HCDR1, a HCDR2, a HCDR3, and the light chain variable region VL comprises a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23 or 27, SEQ ID NO: 24, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, respectively.

[0143] In some embodiments, the anti-CDH17 antibody or antigen-binding fragment thereof of the present application comprises:

[0144] (i) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21, and / or a VL comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; or

[0145] (ii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25, and / or a VL comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 26.

[0146] In some embodiments, an anti-CDH17 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and a light chain variable region, wherein

[0147] (i) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16; or

[0148] (ii) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26.

[0149] In some embodiments, an anti-CDH17 antibody or antigen-binding fragment thereof of the present application further comprises an antibody heavy chain. In some embodiments, an anti-CDH17 antibody or antigen-binding fragment thereof of the present application further comprises an antibody light chain. In some embodiments, an anti-CDH17 antibody or antigen-binding fragment thereof of the present application further comprises a heavy chain and a light chain. In some embodiments, an antibody heavy chain of the present application comprises or consists of the heavy chain variable region and a heavy chain constant region. In some embodiments, an antibody light chain of the present application comprises or consists of the light chain variable region and a light chain constant region. In some embodiments, an antibody of the present application comprises or consists of two heavy chains and two light chains. In some embodiments, an antibody of the present application comprises or consists of two identical heavy chains and two identical light chains.

[0150] In some embodiments, the heavy chain constant region is or is from a (human) IgGl, IgG2, IgG3, or IgG4 heavy chain constant region, e.g., an IgGl heavy chain constant region.

[0151] In some embodiments, the heavy chain constant region comprises

[0152] (i) comprises or consists of an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 55;

[0153] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 55; or

[0154] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 55.

[0155] In some embodiments, the heavy chain constant region comprises or consists of a CH1 and Fc region as described herein.

[0156] In some embodiments, the light chain constant region is or is from a (human) Kappa or Lambda constant region, e.g. a Kappa light chain constant region. In some embodiments, the light chain constant region comprises

[0157] (i) comprises or consists of an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 43 or 56;

[0158] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 43 or 56; or

[0159] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 43 or 56.

[0160] In some embodiments, the antibody or antigen binding fragment thereof of the application comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, the light chain comprises a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region are as defined herein, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43 or 56.

[0161] In some embodiments, the anti-CDH17 antibody or antigen-binding fragment thereof of the present application has one or more of the following properties:

[0162] (i) exhibits the same or similar binding affinity and / or specificity for CDH17 as an anti-CDH17 antibody prepared in accordance with the examples of the present application (e.g., 68A7E3 or Hz68A7E3);

[0163] (ii) inhibits (e.g., competitively inhibits) the binding of an anti-CDH17 antibody prepared in accordance with the examples of the present application (e.g., 68A7E3 or Hz68A7E3) to CDH17;

[0164] (iii) binds to the same or overlapping epitope as an anti-CDH17 antibody prepared in accordance with the examples of the present application (e.g., 68A7E3 or Hz68A7E3);

[0165] (iv) competes with an anti-CDH17 antibody prepared in accordance with the examples of the present application (e.g., 68A7E3 or Hz68A7E3) for binding to CDH17;

[0166] (v) has one or more of the biological properties of an anti-CDH17 antibody prepared in accordance with the examples of the present application (e.g., 68A7E3 or Hz68A7E3).

[0167] In some embodiments, the anti-CDH17 antibody of the present application is an antibody of the IgGl class or an antibody of the IgG2 class or an antibody of the IgG3 class or an antibody of the IgG4 class, preferably an antibody of the IgGl class.

[0168] In some embodiments, the anti-CDH17 antibody is a monoclonal antibody.

[0169] In some embodiments, the anti-CDH17 antibody is humanized. In some embodiments, the humanized anti-CDH17 antibody of the present application can be obtained by a method comprising the steps of:

[0170] 1. determining the CDR loop structure of the heavy chain variable region and the light chain variable region of the parent antibody (e.g., a murine antibody from a hybridoma screen);

[0171] 2. finding the closest homologous sequences for each V / J region of the heavy chain and the light chain in a human germline sequence database;

[0172] 3. screening for the best match of human germline for the heavy chain and the light chain with the minimum amount of back-mutations;

[0173] 4. constructing the CDR regions of the chimeric antibody onto the human scaffold regions;

[0174] V. Using sequence and structural features, determine amino acid positions in the framework region that contribute to maintaining CDR function;

[0175] VI. Perform back-mutations (return to input amino acid type) at sequence positions determined to be important;

[0176] VII. Optimize amino acids at risk sites.

[0177] In some embodiments, the anti-CDH17 antibody is a chimeric antibody.

[0178] In some embodiments, the anti-CDH17 antibody of the application is a full-length antibody.

[0179] In one embodiment, the anti-CDH17 antibody of the application also encompasses antibody fragments (e.g., antigen-binding fragments) thereof, preferably an antibody fragment selected from a Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single domain antibody such as VHH, dAb (domain antibody), diabody, or linear antibody.

[0180] In one embodiment, the anti-CDH17 antibody of the application can also be a bispecific antibody or a multispecific antibody (e.g., a trispecific antibody).

[0181] II-2 Anti-CD16A antibodies or antigen-binding fragments thereof

[0182] One aspect of the application provides a CD16A antibody having a strong binding affinity to CD16A. In some embodiments, the CD16A antibody of the application is suitable for use in constructing an antigen-binding region in a multispecific binding molecule.

[0183] In some embodiments, the anti-CD16A antibody of the application is capable of binding human CD16A and / or cynomolgus CD16A (e.g., CD16A V176) with high affinity, e.g., a K D value of less than or equal to about 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, or 0.6 nM. In some embodiments, the anti-CD16A antibody of the application has a binding K D value of greater than about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, or 0.5 nM. In some embodiments, the anti-CD16A antibody of the application has a binding K DValues are between any two of the above numerical ranges. In some embodiments, the binding affinity of the anti-CD16A antibodies of the present application is determined by biolayer interferometry.

[0184] In some embodiments, the anti-CD16A antibodies of the present application are capable of forming multispecific antibodies, e.g., bispecific antibodies or trispecific antibodies, with other targeting antibodies, e.g., antibodies targeting tumor-associated antigens, to specifically activate the activity of CD16A, e.g., increase its role in ADCC, such as the role of activating NK cells to release cytotoxic molecules to kill the targeted cells, when the targeting antibodies specifically bind to the targeted cells, e.g., tumor cells having the antigen of the targeting antibodies, e.g., tumor-associated antigens.

[0185] In some embodiments, the anti-CD16A antibodies of the present application or antigen-binding fragments thereof are capable of specifically binding to CD16A, e.g., human or cynomolgus CD16A, e.g., specifically binding to CD16A(V176) or CD16A(F176) of human or cynomolgus, or human CD16A.

[0186] In some embodiments, the anti-CD16A antibodies of the present application also encompass multispecific antibodies, e.g., bispecific antibodies or trispecific antibodies, that specifically bind to CD16A and other antigens.

[0187] In some embodiments, the anti-CD16A antibodies of the present application are single domain antibodies, e.g., VHH antibodies.

[0188] In some embodiments, the anti-CD16A single domain antibodies of the present application are VHH antibodies comprising or consisting of a heavy chain variable region, which typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1-3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the “Definitions” section, e.g., the boundaries of the three CDRs in a VHH sequence can be defined by the scheme of Kabat, Chothia, AbM, or IMGT, or a combination thereof.

[0189] In some embodiments, the VHH antibodies against CD16A of the present application comprise

[0190] (i) the three complementarity determining regions (HCDR or VHH CDR) contained in the VH as shown in SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40, or 41, or

[0191] (ii) a sequence which comprises collectively in said three HCDR or VHH CDR regions at least one and not more than 5, 4, 3, 2, or 1 amino acid alteration(s) (preferably amino acid substitution(s), preferably conservative substitution(s)) relative to the sequence of (i);

[0192] wherein said HCDR1, 2, 3 (or VHH CDR1, 2, 3) can be determined according to any of the schemes to determine CDRs, respectively, such as according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, respectively, or a combination thereof;

[0193] For example, the HCDR1 (VHH CDR1 ) sequence is defined according to the Kabat & Chothia combination scheme, the HCDR2 (VHH CDR2) and HCDR3 (VHH CDR3) sequences are defined according to the Kabat scheme, respectively.

[0194] In some embodiments, the VHH antibody against CD16A of the application comprises or consists of a heavy chain variable region comprising

[0195] (i) the three complementarity determining regions (HCDR or VHH CDR) contained in the VH as shown in SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40, or 41, or

[0196] (ii) a sequence which comprises collectively in said three HCDR or VHH CDR regions at least one and not more than 5, 4, 3, 2, or 1 amino acid alteration(s) (preferably amino acid substitution(s), preferably conservative substitution(s)) relative to the sequence of (i);

[0197] wherein said HCDR1, 2, 3 (or VHH CDR1, 2, 3) can be determined according to any of the schemes to determine CDRs, respectively, such as according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, respectively, or a combination thereof;

[0198] For example, the HCDR1 (VHH CDR1 ) sequence is defined according to the Kabat & Chothia combination scheme, the HCDR2 (VHH CDR2) and HCDR3 (VHH CDR3) sequences are defined according to the Kabat scheme, respectively.

[0199] In some embodiments, the anti-CD16A VHH antibody of the application comprises a complementarity determining region (CDR) VHH CDR1, VHH CDR2 and VHH CDR3. In some embodiments, the anti-CD16A VHH of the application comprises or consists of a heavy chain variable region comprising said complementarity determining region (CDR) VHH CDR1, VHH CDR2 and VHH CDR3.

[0200] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 29, 33 or 35, or the VHH CDR1 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 29, 33 or 35.

[0201] In some embodiments, the VHH CDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 13 or 30, or the VHH CDR2 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to an amino acid sequence of SEQ ID NO: 13 or 30.

[0202] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 31, 36 or 42, or the VHH CDR3 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 31, 36 or 42.

[0203] In one embodiment, the anti-CD16A VHH antibody of the application comprises a complementarity determining region (CDR) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0204] (i) the VHH CDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 12, the VHH CDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 13, and the VHH CDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 14 or 36 or 42;

[0205] (ii) the VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or 33, the VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, and the VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 ; or

[0206] (iii) the VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, the VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and the VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36 or 14.

[0207] In one embodiment, the anti-CD16A VHH antibody of the application comprises or consists of a heavy chain variable region, wherein the heavy chain variable region comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0208] (i) the VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, the VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and the VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14 or 36 or 42;

[0209] (ii) the VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or 33, the VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, and the VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 ; or

[0210] (iii) the VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, the VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and the VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36 or 14.

[0211] In some embodiments, the anti-CD16A VHH antibody of the application comprises or consists of a heavy chain variable region, which heavy chain variable region

[0212] (i) comprises or consists of an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40 or 41; or

[0213] (ii) comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40 or 41; or

[0214] (iii) comprises an amino acid sequence which has 1 or more, preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1, amino acid changes, preferably amino acid substitutions, more preferably amino acid conservative substitutions, compared to an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40 or 41, preferably the amino acid changes do not occur in the CDR regions.

[0215] In some embodiments, the anti-CD16A VHH antibody of the application comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40 or 41.

[0216] In one embodiment, the anti-CD16A VHH antibody of the application is a humanized antibody.

[0217] In one embodiment, the anti-CD16A VHH antibody of the application can be comprised in any antibody or multi-specific binding molecule, e.g. a bi-specific antibody or a tri-specific antibody or a fusion protein, as the antigen binding region therein.

[0218] In another aspect of the application, the application also provides an antibody, e.g. a heavy chain antibody or a multi-specific antibody, comprising the anti-CD16A VHH antibody heavy chain variable region of the application.

[0219] In some embodiments, a single domain antibody or VHH of the application, e.g., a camelid VHH or a humanized version thereof, can be linked to a constant region of an antibody or a portion thereof, e.g., an Fc region, to generate a heavy chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the application and an Fc region or constant region at its C-terminus. In some embodiments, the VHH is linked to the Fc via a hinge region or a portion thereof, e.g., a hinge region from an IgG, e.g., a hinge region of IgG1, 2, 3 or 4 or a portion thereof. In some embodiments, the VHH is linked to the Fc via a linker. In some embodiments, the linker comprises (GGGGS)n(SEQ ID NO: 95) or (GGGGG)n(SEQ ID NO: 96), wherein n = any integer between 1-5, e.g., 1, 2, 3, 4 or 5. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45, 46 or 94. In some embodiments, the portion of the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 93.

[0220] In one aspect of the application, the anti-CD16A antibody of the application is a heavy chain antibody against CD16A. In some embodiments, the anti-CD16A heavy chain antibody of the application comprises an anti-CD16A VHH or a heavy chain variable region therein as defined herein, and a heavy chain constant region or an Fc region of a heavy chain constant region, optionally linked via a linker or a hinge region.

[0221] In some embodiments, the heavy chain antibody comprises a constant region that is from or is a constant region of a human or non-human primate (e.g., cynomolgus monkey) antibody, e.g., that is from or is a constant region of human IgG1, human IgG2, human IgG3 or human IgG4.

[0222] In some embodiments, the heavy chain antibody comprises an Fc region that is from or is an Fc region of a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, e.g., a human IgG1, human IgG2, human IgG3 or human IgG4 Fc region, preferably a human IgG1.

[0223] In one embodiment, the heavy chain antibody according to the application can dimerize via the Fc region with another polypeptide chain comprising an Fc region, e.g. another heavy chain antibody of the same or different. Thus, in one embodiment, the present application also provides a homomeric or heteromeric multimeric protein comprising a heavy chain antibody of the present application. In a preferred embodiment, it is preferred that said protein comprises or consists of two identical heavy chain antibody chains paired to form a heavy chain antibody. Thus, in some embodiments, when reference is made to a "heavy chain antibody", a dimer consisting of two heavy chain antibody chains is also encompassed.

[0224] In some embodiments, the anti-CD16A antibody of the application comprises a heavy chain comprising a heavy chain variable region and a Fc region, optionally connected by a hinge region or linker. In some embodiments, the anti-CD16A antibody of the application comprises or consists of a heavy chain comprising or consisting of the heavy chain variable region of an anti-CD16A VHH of the application and a Fc region, optionally the variable region and the Fc region are connected by a hinge region or linker. In some embodiments, the heavy chain variable region of the anti-CD16A VHH is connected to the Fc region by a partial hinge region, e.g. the partial hinge region comprises or consists of EPKSA (SEQ ID NO: 93).

[0225] In some embodiments, the heavy chain antibody of the application comprises or consists of a heavy chain

[0226] (i) comprises or consists of an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 59-67; or

[0227] (ii) comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 59-67; or

[0228] (iii) comprises an amino acid sequence which has 1 or more, preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1, amino acid alterations, preferably amino acid substitutions, more preferably amino acid conservative substitutions, compared to the amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 59-67, preferably the amino acid alterations do not occur in the CDR regions.

[0229] II-3 Multispecific antibodies

[0230] In one aspect of the application, the application relates to multispecific binding molecules that are capable of specifically binding CDH17, and specifically binding one or more other targets or antigens. In some embodiments, the other antigen is EGFR. In some embodiments, the other antigen is CD16A and / or EGFR, e.g., CD16A and EGFR.

[0231] In one aspect of the application, the application relates to multispecific binding molecules that are capable of specifically binding CD16A, and specifically binding one or more other targets or antigens. In some embodiments, the other antigen is CDH17 and / or EGFR.

[0232] In some embodiments, the multispecific binding molecule is a multispecific antibody, e.g., a bispecific antibody or a trispecific antibody.

[0233] In some embodiments, the antibody of the application is a bispecific antibody, e.g., an anti-CDH17 x EGFR bispecific antibody, that specifically binds CDH17 (e.g., human CDH17) and EGFR (e.g., human EGFR).

[0234] In some embodiments, the antibody of the application is a trispecific antibody, e.g., a trispecific antibody that specifically binds CDH17 (e.g., human CDH17), EGFR (e.g., human EGFR), and CD16A (e.g., human CD16A).

[0235] Thus, one aspect of the application relates to a bispecific antibody comprising

[0236] a first antigen binding region and a second antigen binding region, wherein the first antigen binding region specifically binds EGFR (e.g., human EGFR) and the second antigen binding region specifically CDH17 (e.g., human CDH17).

[0237] Thus, one aspect of the application relates to a trispecific antibody comprising

[0238] a first antigen binding region, a second antigen binding region, and a third antigen binding region, wherein the first antigen binding region specifically binds EGFR (e.g., human EGFR), the second antigen binding region specifically binds CDH17 (e.g., human CDH17), and the third antigen binding region specifically binds CD16A (e.g., human CD16A).

[0239] The first antigen binding region of the multispecific antibody suitable for use in the application can comprise or consist of an antibody or antigen binding fragment thereof that is specific for EGFR, including but not limited to, for example, a full-length antibody, a half-antibody, a Fab, a Fab’, a Fab’-SH, a Fv, a single-chain antibody (e.g., scFv), a (Fab’)2, a single domain antibody (e.g., VHH), a dAb (domain antibody), a heavy chain antibody, or a linear antibody, etc., that is specific for EGFR.

[0240] The second antigen binding region of the multispecific antibody suitable for use in the application can comprise or consist of an anti-CDH17 antibody or antigen binding fragment thereof of the application (an anti-CDH17 antibody or antigen binding fragment thereof of the application described in Section II-1), including but not limited to, for example, a full-length antibody, a half-antibody, a Fab, a Fab’, a Fab’-SH, a Fv, a single-chain antibody (e.g., scFv), a (Fab’)2, a single domain antibody (e.g., VHH), a dAb (domain antibody), a heavy chain antibody, or a linear antibody, etc., that is specific for CDH17.

[0241] The third antigen binding region of the multispecific antibody suitable for use in the application can comprise or consist of an antibody or antigen binding fragment thereof that is specific for CD16A, including but not limited to, for example, a full-length antibody, a half-antibody, a Fab, a Fab’, a Fab’-SH, a Fv, a single-chain antibody (e.g., scFv), a (Fab’)2, a single domain antibody (e.g., VHH), a dAb (domain antibody), a heavy chain antibody, or a linear antibody, etc., that is specific for CD16A.

[0242] In some embodiments, the third antigen binding region is derived from an anti-CD16A antibody or antigen binding fragment thereof described herein (an anti-CD16A antibody or antigen binding fragment thereof of the application described in Section II-I1), e.g., is an anti-CD16A VHH antibody. In some embodiments, the third antigen binding region of the multispecific antibody suitable for use in the application can comprise 3 HCDRs of an anti-CD16A VHH antibody described herein. In some embodiments, the third antigen binding region of the multispecific antibody suitable for use in the application can comprise an anti-CD16A VHH antibody described herein.

[0243] In some embodiments, the multispecific antibody of the application is an IgG-like multispecific antibody. In some embodiments, the IgG-like multispecific antibody of the application refers to a multispecific antibody comprising an Fc dimer, e.g., a heterodimeric Fc scaffold. Thus, in some embodiments, the multispecific antibody of the application comprises an Fc dimer, e.g., a heterodimeric Fc scaffold.

[0244] In one embodiment, the multispecific antibody can comprise one or more first antigen binding regions. In one embodiment, the multispecific antibody can comprise one or more second antigen binding regions. In one embodiment, the multispecific antibody can comprise one or more third antigen binding regions. In one embodiment, the bispecific antibody of the application comprises one first antigen binding region and one second antigen binding region. In one embodiment, the trispecific antibody of the application comprises one first antigen binding region, one second antigen binding region, and two third antigen binding regions.

[0245] The components of the multispecific binding molecules of the application are described in detail below. It will be understood by those skilled in the art that any combination of features of any of these components is within the scope of the application, unless the context clearly indicates otherwise. Also, it will be understood by those skilled in the art that the multispecific binding molecules of the application, including any form of antibody, can comprise any such combination of features, unless the context clearly indicates otherwise.

[0246] II-3-1 First antigen binding region

[0247] In some embodiments, the first antigen binding region specifically binds EGFR.

[0248] In some embodiments, the first antigen binding region is an antigen binding region that specifically binds EGFR. In some embodiments, the first antigen binding region that specifically binds EGFR is derived from an antibody that specifically binds EGFR, e.g., an antibody that specifically binds EGFR disclosed in US20100008929A1 (incorporated herein in its entirety), e.g., Zalutumumab disclosed therein.

[0249] In some embodiments, the first antigen binding region that specifically binds EGFR comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, or LCDR3, of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises a heavy chain variable region of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises a light chain variable region of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein). In some embodiments, the first antigen binding region comprises or consists of a Fab of a known antibody that specifically binds EGFR (e.g., an EGFR antibody disclosed in US20100008929A1, e.g., Zalutumumab disclosed therein).

[0250] In some embodiments, the first antigen binding region that specifically binds EGFR comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO: 7. In some embodiments, the first antigen binding region that specifically binds EGFR comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of a light chain variable region as set forth in SEQ ID NO: 2. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can each be determined according to any scheme for determining CDRs, e.g., according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, or combinations thereof, respectively; e.g., the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are determined according to the Kabat scheme, respectively, and the LCDR1, 2, and 3 are determined according to the Kabat scheme, respectively.

[0251] In some embodiments, the HCDR1 of the first antigen binding region that specifically binds EGFR comprises or consists of the amino acid sequence of SEQ ID NO: 8; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0252] In some embodiments, the first antigen binding region that specifically binds EGFR comprises a VH, wherein the VH comprises

[0253] (a) 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; or

[0254] (b) the amino acid sequence set forth in SEQ ID NO: 7, or consists of the same.

[0255] In some embodiments, the first antigen binding region that specifically binds EGFR comprises a VL, wherein the VL comprises

[0256] (a) 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; and

[0257] (b) the amino acid sequence set forth in SEQ ID NO: 2 or consists of the amino acid sequence.

[0258] In some embodiments, the first antigen binding region that specifically binds EGFR comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3 and 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0259] In some embodiments, the first antigen binding region that specifically binds EGFR comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 7; and / or the VL comprises or consists of the sequence set forth in SEQ ID NO: 2.

[0260] In some embodiments, the first antigen binding region that specifically binds EGFR is a Fab fragment.

[0261] II-3-2 Second antigen binding region

[0262] In some embodiments, the second antigen binding region specifically binds CDH17, e.g., human CDH17.

[0263] In some embodiments, the second antigen binding region is derived from an anti-CDH17 antibody or antigen binding fragment thereof described herein, e.g., an anti-CDH17 antibody or antigen binding fragment thereof as shown in II-1, e.g., a Fab against CDH17. In some embodiments, the second antigen binding region that specifically binds CDH17 comprises 1, 2, 3, 4, 5, or 6 CDRs of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises 1, 2, or 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises a heavy chain variable region of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises a light chain variable region of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises a heavy chain variable region and a light chain variable region of an anti-CDH17 antibody described herein. In some embodiments, the second antigen binding region comprises or consists of a Fab of an anti-CDH17 antibody described herein.

[0264] In some embodiments, the second antigen binding region is an antigen binding region that specifically binds CDH17. In some embodiments, the second antigen binding region that specifically binds CDH17 is derived from an antibody that specifically binds CDH17, e.g., an antibody that specifically binds CDH17 disclosed in WO2018115231A2 (incorporated herein in its entirety).

[0265] In some embodiments, the second antigen binding region that specifically binds CDH17 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises 1, 2, or 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises a heavy chain variable region of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises a light chain variable region of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2). In some embodiments, the second antigen binding region comprises or consists of a Fab of a known antibody that specifically binds CDH17 (e.g., a CDH17 antibody disclosed in WO2018115231A2).

[0266] In some embodiments, the second antigen binding region that specifically binds CDH17 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 88. In some embodiments, the second antigen binding region that specifically binds CDH17 comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 89. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can each be determined according to any scheme for determining CDRs, e.g., according to the scheme of Kabat, AbM, Chothia, Contact, or IMGT, or combinations thereof, respectively; e.g., the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are determined according to the Kabat scheme, respectively, and the LCDR1, 2, and 3 are determined according to the Kabat scheme, respectively.

[0267] In some embodiments, the HCDR1 of the second antigen binding region that specifically binds CDH17 comprises or consists of the amino acid sequence of SEQ ID NO: 97; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 98, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 99; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 100, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 101, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 102.

[0268] In some embodiments, the second antigen binding region that specifically binds CDH17 comprises a VH, wherein the VH comprises

[0269] (a) 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 97; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 98, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 99; or

[0270] (b) the amino acid sequence set forth in SEQ ID NO: 88.

[0271] In some embodiments, the second antigen binding region that specifically binds CDH17 comprises a VL, wherein the VL comprises

[0272] (a) 3 complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3 from a heavy chain variable region, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 97; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 98, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 99; and 3 complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3 from a light chain variable region, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 100; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 101, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 102.

[0273] (b) the amino acid sequence set forth in SEQ ID NO: 89, or a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 89.

[0274] In some embodiments, the second antigen binding region that specifically binds CDH17 comprises 3 complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3 from a heavy chain variable region, and 3 complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3 from a light chain variable region, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 97; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 98, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 99, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 100, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 101, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 102.

[0275] In some embodiments, the second antigen binding region that specifically binds CDH17 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 88; and / or the VL comprises or consists of the sequence set forth in SEQ ID NO: 89.

[0276] In some embodiments, the second antigen binding region that specifically binds CDH17 is a Fab fragment.

[0277] II-3-3 Fab fragment

[0278] In some embodiments, the first and / or second antigen binding region described herein is a Fab fragment. A Fab fragment suitable for use as an antigen binding region of a multispecific antibody, e.g., a trispecific antibody, consists of two polypeptide chains comprising the VH, CH1, VL and CL domains of an antibody, wherein the VH pairs with the VL and the CH1 pairs with the CL to form an antigen binding region. In some embodiments, in a Fab, one chain comprises or consists of VH and CH1 (i.e., VH-CH1 ) from the N- to C-terminus, and the other chain comprises or consists of VL and CL (i.e., VL-CL) from the N- to C-terminus. In some embodiments, in a multispecific antibody of the application, a Fab can be linked at the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising the VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and can be linked to the antibody Fc domain via the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the linkage is a direct linkage, or via a linker. In the present context, a Fab chain comprising or consisting of VH-CH1 is also referred to as Fab heavy chain, and a Fab chain comprising or consisting of VL-CL is also referred to as Fab light chain.

[0279] In some embodiments, the CH1 is or is from a CH1 of IgG1, IgG2, IgG3 or IgG4, preferably a CH1 of IgG1. In some embodiments, the CH1 is or is from a CH1 of IgG1, IgG2, IgG3 or IgG4, preferably a CH1 of IgG1, which has been modified to reduce effector function, e.g., Fc receptor binding and / or complement binding. In some embodiments, the CH1 is or is from a CH1 of IgG1, IgG2, IgG3 or IgG4, preferably a CH1 of IgG1, which has been modified to reduce effector function, e.g., Fc receptor binding and / or complement binding, and which has been modified to increase antigen binding, e.g., to increase the off-rate of the antigen binding region from the antigen.

[0280] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 44;

[0281] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 44; or

[0282] (iii) comprises or consists of an amino acid sequence which has 1 or more, preferably not more than 10 or 10, more preferably not more than 5, 4, 3, 2, 1, amino acid alterations, preferably amino acid substitutions, more preferably amino acid conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 44.

[0283] In some embodiments, the CL is or is from a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, the CL is or is from a Kappa light chain constant region. In some embodiments, the CL is or is from a Kappa light chain constant region which has been modified to reduce effector function, e.g., Fc receptor binding and / or complement binding. In some embodiments, the CL is or is from a Kappa light chain constant region which has been modified to reduce effector function, e.g., Fc receptor binding and / or complement binding, and which has been modified to increase antigen binding, e.g., to increase the off-rate of the antigen binding region from the antigen.

[0284] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 43 or 56;

[0285] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 43 or 56; or

[0286] (iii) comprises or consists of an amino acid sequence which has 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 43 or 56.

[0287] In some embodiments, the first antigen binding region is a Fab that specifically binds EGFR, wherein the Fab fragment is from an anti-EGFR antibody comprising a heavy chain variable region VH and a light chain variable region VL of an anti-EGFR antibody (e.g. a heavy chain variable region VH and a light chain variable region VL as defined in II-3-1). In some embodiments, the Fab heavy chain of the Fab that specifically binds EGFR antibody as the first antigen binding region comprises or consists of a VH and a CH1, wherein the VH is a VH of an anti-EGFR antibody (e.g. a VH of the first antigen binding region as defined herein). In some embodiments, the Fab light chain of the Fab that specifically binds EGFR as the first antigen binding region comprises or consists of a VL and a CL, wherein the VL is a VL of an anti-EGFR antibody (e.g. a VL of the first antigen binding region as defined herein). In some embodiments, the first antigen binding region comprises a CH1 from an IgGl, and / or a Kappa light chain constant region.

[0288] In some embodiments, the Fab heavy chain that specifically binds EGFR comprises

[0289] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 49;

[0290] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 49; or

[0291] (iii) comprises or consists of an amino acid sequence having 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 49.

[0292] In some embodiments, the Fab light chain that specifically binds to EGFR comprises

[0293] (i) comprises or consists of an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1 ;

[0294] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 1 ; or

[0295] (iii) comprises or consists of an amino acid sequence having 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1.

[0296] In some embodiments, the Fab that specifically binds to EGFR comprises or consists of a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the Fab that specifically binds to EGFR comprises or consists of one Fab heavy chain and one Fab light chain, wherein the Fab heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0297] In some embodiments, the second antigen binding region is a Fab that specifically binds to CDH17, wherein the Fab fragment is derived from an anti-CDH17 antibody of the application (e.g., an anti-CDH17 antibody of the application or antigen binding fragment thereof as described in II-1), comprising a heavy chain variable region VH and a light chain variable region VL of an anti-CDH17 antibody (e.g., a VH and VL as defined in II-1, or a VH and VL as defined in II-3-2).

[0298] In some embodiments, the Fab that is the second antigen binding region comprises the VH or VL of an anti-CDH17 antibody described herein, or comprises the VH and VL of an anti-CDH17 antibody described herein.

[0299] In some embodiments, the Fab that is the second antigen binding region comprises the VH or VL of a second antigen binding region described herein, or comprises the VH and VL of a second antigen binding region described herein.

[0300] In some embodiments, the Fab heavy chain of a CDH17 antibody that specifically binds comprises or consists of a VH and a CH1, wherein the VH is a VH of a second antigen binding region described herein. In some embodiments, the Fab light chain of a CDH17 antibody that specifically binds comprises or consists of a VL and a CL, wherein the VL is a VL of a second antigen binding region described herein. In some embodiments, the second antigen binding region comprises a CH1 from an IgGl, and / or a Lambda light chain constant region.

[0301] In some embodiments, the Fab heavy chain of a CDH17 antibody that specifically binds comprises

[0302] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 50 or 90;

[0303] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 50 or 90; or

[0304] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 50 or 90.

[0305] In some embodiments, the Fab light chain of a CDH17 antibody that specifically binds comprises

[0306] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15 or 92;

[0307] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 15 or 92; or

[0308] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alteration(s) (preferably amino acid substitution(s), more preferably amino acid conservative substitution(s)) compared to the amino acid sequence of SEQ ID NO: 15 or 92.

[0309] In some embodiments, the Fab that specifically binds CDH17 comprises or consists of a Fab heavy chain and a Fab light chain. In some embodiments, the Fab that specifically binds CDH17 comprises or consists of one Fab heavy chain and one Fab light chain. In some embodiments, the Fab heavy chain that specifically binds CDH17 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the Fab heavy chain that specifically binds CDH17 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 92.

[0310] In some embodiments, the light chain constant region in the Fab light chain of the first antigen binding region is the same or different from the light chain constant region of the Fab light chain of the second antigen binding region. In some embodiments, the light chain constant region of the Fab light chain of the first and second antigen binding region are both Kappa light chain constant region or Lambda light chain constant region.

[0311] II-3-4 Fc region

[0312] In some embodiments, one or more target binding regions (antigen binding regions) of the multispecific antibody (e.g., bispecific antibody or trispecific antibody) of the present application further comprises a Fc region (e.g., a first Fc region and a second Fc region), wherein the Fc regions comprised can be the same or different.

[0313] In some embodiments, the first Fc region and the second Fc region are different, which are capable of dimerizing to form a heterodimeric Fc scaffold.

[0314] In the present context, Fc region refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and can include native sequence Fc regions and variant Fc regions. Native sequence Fc region encompasses the Fc regions of the various human immunoglobulin isotypes, such as IgG, IgA, IgE, and IgD, as well as the Fc region of the various allotypes of these isotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present application comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region can also carry an IgG hinge region or a partial IgG hinge region at the N-terminus, e.g., an IgGl hinge region or a partial IgGl hinge region, e.g., the sequence from D221 to P230 according to EU numbering. Mutations can be contained in the hinge region.

[0315] Unless otherwise indicated herein, numbering of amino acid residues in the Fc region is according to the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0316] In some embodiments, the Fc region is or is from a human IgG Fc, e.g., is a human IgGl Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or 58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto.

[0317] As understood by the skilled person, in order to facilitate the formation of the multispecific antibody of the present application as a heterodimer, the Fc region comprised by the multispecific antibody of the present application can comprise mutations that facilitate heterodimerization of the first Fc region with the second Fc region. In one embodiment, mutations are introduced in the CH3 region of both Fc regions.

[0318] Methods to promote Fc region heterodimerization are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary fashion such that each CH3 region (or heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the other, complementary engineered CH3 region (such that the CH3 regions of the first and second Fc region heterodimerize and no homodimers between two first CH3 regions or two second CH3 regions are formed). For example, based on the Knob-into-Hole technology, respective Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region. This technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0319] Respective mutations can also be introduced in the first CH3 region of the first Fc region and the second CH3 region of the second Fc region based on the Innobody technology. This technology is described, for example, in WO2022143912A1 (which patent is incorporated herein in its entirety).

[0320] In a particular embodiment, the first CH3 region comprises a S364R / K mutation (preferably S364R), and optionally one or more further mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S), and optionally one or more further mutations. In some embodiments, the first CH3 region comprises a S364R / K mutation, and the second CH3 region comprises a K370S / T / A / V mutation. In some embodiments, the first CH3 region comprises a S364R mutation, and the second CH3 region comprises a K370S mutation.

[0321] In some embodiments, the first CH3 region comprises S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises K370S / T / A / V (preferably K370S) mutation and K409D / E (preferably K409D) mutation and / or Y349T / S / A / V mutation. In some embodiments, the first CH3 region comprises S364R / K + D399K / R, and the second CH3 region comprises K370S / T / A / V + Y349T / S / A / V. In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T. In some embodiments, the first CH3 region further comprises E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further comprises K409D / E (preferably K409D), Q347D / E (preferably Q347D), and / or T350V / A (preferably T350V).

[0322] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region further comprises E357N, and the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, respectively, or both comprise T350V.

[0323] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region comprises S364R + D399K + E357N, and the second CH3 region comprises K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region comprises S364R + D399K + E357N + T350V, and the second CH3 region comprises K370S + Y349T + K409D + Q347D + T350V.

[0324] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E) and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411 R / K (preferably T411 R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D + T411 R / K and the second CH3 region comprises D399K / R + K370T / S / A / V. In some embodiments, the CH3 region comprises K409E + T411 R and the second CH3 region comprises D399K + K370T.

[0325] In some specific embodiments, the first and second CH3 regions have the following 5 combinations of mutations:

[0326] In one embodiment, the CH3 of one Fc region comprises S364R and D399K mutations and the CH3 mutations of the other Fc region comprise Y349T, K370S and K409D mutations.

[0327] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0328] The Fc region in the binding molecules of the application, e.g., antibodies, can also be mutated to obtain desired properties. Mutations to the Fc region are known in the art.

[0329] In one embodiment, the Fc region is modified in a property of an effector function of the Fc region, e.g., a complement activation function of the Fc region. In one embodiment, the effector function has been reduced or eliminated relative to a wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: use of an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0330] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, e.g., reduced or eliminated ADCC or ADCP or CDC effector function, e.g., comprising mutations that achieve the above functions.

[0331] As those skilled in the art will understand, in accordance with the intended use of the binding molecules, such as antibody molecules, according to the present invention, the binding molecules, such as antibody molecules, may also include modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region contains mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has an L234A / L235A mutation (LALA mutation) that reduces binding to the Fcγ receptor. In yet another preferred embodiment, the Fc fragment may have mutations that result in an increased serum half-life, such as mutations that improve the binding of the Fc fragment to FcRn.

[0332] Therefore, in one specific embodiment, the antibody of the present invention, such as a multispecific antibody, comprises two heterodimerized Fc regions, one Fc-region polypeptide comprising the mutations S364R and D399K, and the other Fc-region polypeptide comprising the mutations Y349T, K370S, and K409D. In some embodiments, the Fc regions further comprise L234A / L235A mutations, respectively.

[0333] Therefore, in one specific embodiment, the antibody of the present invention, such as a multispecific antibody, comprises two heterodimerized Fc regions, wherein...

[0334] One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:47 or 86, while the other Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO:48 or 87.

[0335] One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:47, while the other Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO:48; or

[0336] One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:86, while the other Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO:87.

[0337] In one specific embodiment, the antibody of the present invention, such as a multispecific antibody, comprises heterodimerized two Fc regions, wherein...

[0338] One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47 or 86, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:48 or 87.

[0339] One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:48; or

[0340] One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:86, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87.

[0341] Therefore, in one specific embodiment, the two Fc regions of the binding molecule of the present invention, such as an antibody like a multispecific antibody, are heterodimerized, wherein...

[0342] a) One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:86 and contains the mutations S364R and D399K, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87 and contains the mutations Y349T, K370S, and K409D; or

[0343] b) One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47 and contains the mutations S364R and D399K and L234A / L235A mutations, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:48 and contains the mutations Y349T, K370S, and K409D and L234A / L235A mutations.

[0344] II-3-5 Exemplary Bispecific Antibody Structure

[0345] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region (e.g., the first antigen-binding region described in Section II-3-1) that specifically binds to a first antigen as described herein, and a second antigen-binding region (e.g., the second antigen-binding region described in Section II-3-2) that specifically binds to a second antigen as described herein, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab. In some embodiments, the bispecific antibody of the present invention comprises a first Fab and a second Fab.

[0346] In some embodiments, the first Fab is connected at the C-terminus of its CH1 region to the N-terminus of the first Fc region (via or without a connector, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 region to the N-terminus of the second Fc region (via or without a connector, such as a hinge region). In a specific embodiment, the first Fab and the first Fc region constitute a first hapten that binds to a first antigen, the second Fab and the second Fc region constitute a second hapten that binds to a second antigen, and the first hapten and the second hapten constitute the bispecific antibody of the present invention.

[0347] In one specific embodiment, the bispecific antibody is an IgG-like antibody having the configuration shown as P001, P007, or P008 in Figure 5.

[0348] In one specific implementation, the first Fc region and the second Fc region contain Innobody mutations, as well as optional mutations that reduce the function of effectors mediated by the Fc region or reduce binding to the Fcγ receptor.

[0349] In one embodiment, the bispecific antibody comprises or consists of the following chains:

[0350] Heavy chain A: from the N end to the C end, it contains or consists of the following: first Fab heavy chain variable region - heavy chain constant region CH1 - first Fc region;

[0351] Light chain a: from the N end to the C end, it includes or consists of the following: the light chain variable region of the first Fab - the light chain constant region;

[0352] Heavy chain B: From the N end to the C end, it contains or consists of the following: second Fab heavy chain variable region - heavy chain constant region CH1 - second Fc region;

[0353] Light chain b: From the N end to the C end, it includes the following components: the light chain variable region of the second Fab - the light chain constant region;

[0354] The first Fab specifically binds to the first antigen, and the second Fab specifically binds to the second antigen.

[0355] In some specific implementations, different units are connected by connectors, for example, in heavy chain A and / or heavy chain B, different units are connected by connectors.

[0356] The connectors suitable for use in this invention include glycine connectors, such as (G). n , where n = any integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5 or 10. In some embodiments, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO: 45 or 46.

[0357] In some embodiments, the Fab heavy chain variable region and the heavy chain constant region CH1 constitute the Fab heavy chain, wherein CH1 is defined as in II-3-3. In some embodiments, the Fab light chain variable region and the light chain constant region constitute the Fab light chain, wherein the light chain constant region is defined as in II-3-3.

[0358] In one specific implementation, the first Fab is an EGFR-resistant Fab (such as the EGFR-specific Fab defined in II-3-3), and the second Fab is the CDH17-resistant Fab of the present invention (such as the CDH17-specific Fab defined in II-3-3), or vice versa.

[0359] In one specific embodiment, the bispecific antibody of the present invention specifically binds to EGFR and CDH17, and comprises or consists of the following:

[0360] Heavy chain A: From the N-terminus to the C-terminus, it contains or consists of the following: Fab heavy chain variable region that specifically binds to EGFR - heavy chain constant region CH1 - first Fc region;

[0361] Light chain a: from the N-terminus to the C-terminus, it contains or consists of the following: a light chain variable region of Fab that specifically binds to EGFR - a light chain constant region;

[0362] Heavy chain B: From the N-terminus to the C-terminus, it contains or consists of the following: a CDH17-specifically binding Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region;

[0363] Light chain b: Composed of the following components from the N-terminus to the C-terminus: light chain variable region that specifically binds to CDH17 - light chain constant region;

[0364] The units may be connected via or without connectors (e.g., as defined herein).

[0365] Optionally, the heavy chain variable region and light chain variable region of the Fab that specifically binds to EGFR are as defined herein, for example as defined in Section II-3-1, or the Fab that specifically binds to EGFR is as defined in II-3-3;

[0366] Optionally, the heavy chain variable region and light chain variable region of a CDH17-specific Fab are as defined herein, for example as defined in Section II-1 or Section II-3-2, or the CDH17-specific Fab is as defined in II-3-3.

[0367] Optionally, the heavy chain constant region CH1 and the light chain constant region are as defined in Section II-3-3, respectively; and / or

[0368] Optionally, the first Fc region and the second Fc region are as defined in Section II-3-4.

[0369] In some implementations, the first Fc region contains S364R and D399K mutations, and the second Fc region contains Y349T, K370S, and K409D mutations; optionally, the first Fc region and the second Fc region also contain L234A / L235A mutations, respectively.

[0370] In some implementations, the light chain constant region in light chain a may be the same as or different from the light chain constant region in light chain b. For example, the light chain constant regions in light chains a and b are Kappa light chain constant regions as defined herein.

[0371] In some specific embodiments, the bispecific antibody that specifically binds to EGFR and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0372] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:70 or 74, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0373] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0374] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO: 91, 68, or 80, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0375] The light chain b comprises the amino acid sequence shown in SEQ ID NO: 92 or 15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0376] In some specific embodiments, the bispecific antibody that specifically binds to EGFR and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0377] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:70, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0378] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0379] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:91, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0380] The light chain b comprises the amino acid sequence shown in SEQ ID NO:92, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0381] In some specific embodiments, the bispecific antibody that specifically binds to EGFR and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0382] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:70, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0383] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0384] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0385] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0386] In some specific embodiments, the bispecific antibody that specifically binds to EGFR and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0387] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:74, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0388] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0389] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0390] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0391] In some embodiments, the bispecific antibody of the present invention comprises any one or more domains (e.g., the domains shown in Table 9 or the domains shown in Table 10 of P001) of the Zalu / CDH17v1 or Zalu / CDH17 bispecific antibody shown in Example 8 or Example 9.

[0392] II-3-6 Exemplary Trispecific Antibody Structure

[0393] In some embodiments, the trispecific antibody of the present invention comprises a first antigen-binding region that specifically binds to a first antigen (e.g., the first antigen-binding region described in Section II-3-1), a second antigen-binding region that specifically binds to a second antigen (e.g., the second antigen-binding region described in Section II-3-2), and a third antigen-binding region that specifically binds to a third antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, and the third antigen-binding region comprises a VHH (e.g., the VHH described in Section II-2).

[0394] In some embodiments, the trispecific antibody comprises a first Fab, a second Fab, and two VHHs. In some embodiments, the two VHHs are respectively attached to the N-terminus of the Fab heavy chain of the first Fab and the second Fab, or respectively inserted between the C-terminus and the Fc region of the Fab heavy chain of the first Fab and the second Fab, or respectively attached to the N-terminus or C-terminus of the Fab light chain of the first Fab and the second Fab, or respectively attached to the C-terminus of the first Fc region and the second Fc region. In some embodiments, the two VHHs may be the same or different, for example, identical.

[0395] In some embodiments, the first Fab is linked to a third antigen-binding region VHH (first VHH) at the C-terminus of its CH1 region, and the third antigen-binding region VHH is linked to the N-terminus of the first Fc region (via or without a linker). The second Fab is linked to a third antigen-binding region VHH (second VHH) at the C-terminus of its CH1 region, and the third antigen-binding region VHH is linked to the N-terminus of the second Fc region (via or without a linker). In a specific embodiment, the first Fab, VHH, and the first Fc region constitute a first hapten antibody that binds to a first antigen, the second Fab, VHH, and the second Fc region constitute a second hapten antibody that binds to a second antigen, and the first and second haptens constitute the trispecific antibody of the present invention.

[0396] In one specific implementation, the trispecific antibody is an IgG-like antibody having the configuration shown in Figure 5 as P002-P006.

[0397] In one specific implementation, the first Fc region and the second Fc region contain Innobody mutations, as well as optional mutations that reduce the function of Fc region-mediated effectors or reduce binding to the Fcγ receptor, such as LALA mutations (L234A / L235A mutations).

[0398] In one embodiment, the trispecific antibody comprises or is composed of the following:

[0399] Heavy chain A: From the N end to the C end, it includes or consists of the following: first Fab heavy chain variable region - heavy chain constant region CH1 - first VHH - first Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first VHH, and / or the first VHH is connected at its C end to the N end of the first Fc region via or without a connector.

[0400] Light chain a: from the N end to the C end, it includes or consists of the following: the light chain variable region of the first Fab - the light chain constant region;

[0401] Heavy chain B: From the N end to the C end, it includes or consists of the following: second Fab heavy chain variable region - heavy chain constant region CH1 - second VHH - second Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the second VHH, and / or the second VHH is connected at its C end to the N end of the second Fc region via or without a connector.

[0402] Light chain b: From the N end to the C end, it includes the following components: the light chain variable region of the second Fab - the light chain constant region;

[0403] The first Fab specifically binds to the first antigen, and the second Fab specifically binds to the second antigen;

[0404] The first VHH and the second VHH both specifically bind to the third antigen, and they may be the same or different, but preferably the same.

[0405] In some specific implementations, different units are connected by connectors, for example, in heavy chain A and / or heavy chain B, different units are connected by connectors.

[0406] In one specific embodiment, the first or second VHH is connected at its N-terminus to the C-terminus of the first or second Fab heavy chain via connector 1, and the first or second VHH is connected at its C-terminus to the N-terminus of the first or second Fc region via connector 2. In some embodiments, connector 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:45, and / or connector 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:46.

[0407] In one embodiment, the trispecific antibody comprises or is composed of the following:

[0408] Heavy chain A: from the N end to the C end, it contains or consists of the following: first Fab heavy chain variable region - heavy chain constant region CH1 - first Fc region;

[0409] Light chain a: from the N end to the C end, it includes or consists of the following: the light chain variable region of the first Fab - the light chain constant region;

[0410] Heavy chain B: From the N end to the C end, it contains or consists of the following: second Fab heavy chain variable region - heavy chain constant region CH1 - second Fc region;

[0411] Light chain b: From the N end to the C end, it includes the following components: the light chain variable region of the second Fab - the light chain constant region;

[0412] The first Fab specifically binds to the first antigen, and the second Fab specifically binds to the second antigen;

[0413] The heavy chains A and B further comprise a first VHH and a second VHH, respectively, wherein the C-ends of the first VHH and the second VHH are connected, with or without a connector, to the N-ends of the first Fab heavy chain variable region and the second Fab heavy chain variable region; or the N-ends of the first VHH and the second VHH are connected, with or without a connector, to the C-ends of the first Fc region and the second Fc region; or

[0414] The light chains a and b further include a first VHH and a second VHH, wherein the C-ends of the first VHH and the second VHH are connected to the N-ends of the first Fab light chain variable region and the second Fab light chain variable region via or without a connector, or wherein the N-ends of the first VHH and the second VHH are connected to the C-ends of the first Fab light chain variable region and the second Fab light chain variable region via or without a connector.

[0415] The first VHH and the second VHH both specifically bind to the third antigen; they may be the same or different, but preferably the same.

[0416] Optionally, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO:45.

[0417] The connectors suitable for use in this invention include glycine connectors, such as (G). n , where n = any integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5 or 10. In some embodiments, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO: 45 or 46.

[0418] In some embodiments, the Fab heavy chain variable region and the heavy chain constant region CH1 constitute the Fab heavy chain, wherein CH1 is defined as in II-3-3. In some embodiments, the Fab light chain variable region and the light chain constant region constitute the Fab light chain, wherein the light chain constant region is defined as in II-3-3.

[0419] In one specific implementation, the first Fab is an EGFR-resistant Fab (e.g., the Fab described herein, such as the EGFR-specific Fab as defined in II-3-3), and the second Fab is an EGFR-resistant Fab (e.g., the Fab described herein, such as the EGFR-specific Fab as defined in II-3-3), or vice versa. Optionally, the first and second VHHs are CD16A-resistant VHHs of the present invention (e.g., CD16A-resistant VHHs as defined in Section II-2).

[0420] In one specific embodiment, the trispecific antibody of the present invention specifically binds to EGFR, CD16A and CDH17 and CD16A, and comprises or consists of the following:

[0421] Heavy chain A: From the N-terminus to the C-terminus, it comprises or consists of the following: a Fab heavy chain variable region specifically binding to EGFR - a heavy chain constant region CH1 - a first VHH specifically binding to CD16A - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first VHH, and / or the first VHH is connected at its C-terminus to the N-terminus of the first Fc region via or not via a connector.

[0422] Light chain a: from the N-terminus to the C-terminus, it contains or consists of the following: a light chain variable region of Fab that specifically binds to EGFR - a light chain constant region;

[0423] Heavy chain B: From the N-terminus to the C-terminus, it comprises or consists of the following: a Fab heavy chain variable region specifically binding to CDH17 - a heavy chain constant region CH1 - a second VHH specifically binding to CD16A - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second VHH, and / or the second VHH is connected at its C-terminus to the N-terminus of the second Fc region via or not via a connector;

[0424] Light chain b: From the N-terminus to the C-terminus, it contains or consists of the following: a light chain variable region that specifically binds to CDH17 - a light chain constant region;

[0425] The first VHH and the second VHH can be the same or different, preferably the same, for example, as defined in Section II-2 respectively;

[0426] Optionally, the heavy chain variable region and light chain variable region of the Fab that specifically binds to EGFR are as defined herein, for example as defined in Section II-3-1, or the Fab that specifically binds to EGFR is as defined in II-3-3;

[0427] Optionally, the heavy chain variable region and light chain variable region of a CDH17-specific Fab are as defined herein, for example as defined in Section II-1 or Section II-3-2, or the CDH17-specific Fab is as defined in II-3-3.

[0428] Optionally, the heavy chain constant region CH1 and the light chain constant region are as defined in Section II-3-3, respectively; and / or

[0429] Optionally, the first Fc region and the second Fc region are as defined in Section II-3-4.

[0430] In one specific embodiment, the trispecific antibody of the present invention specifically binds to EGFR, CD16A and CDH17 and CD16A, and comprises or consists of the following:

[0431] Heavy chain A: From the N-terminus to the C-terminus, it contains or consists of the following: Fab heavy chain variable region that specifically binds to EGFR - heavy chain constant region CH1 - first Fc region;

[0432] Light chain a: from the N-terminus to the C-terminus, it contains or consists of the following: a light chain variable region of Fab that specifically binds to EGFR - a light chain constant region;

[0433] Heavy chain B: From the N-terminus to the C-terminus, it contains or consists of the following: a CDH17-specifically binding Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region;

[0434] Light chain b: Composed of the following components from the N-terminus to the C-terminus: light chain variable region that specifically binds to CDH17 - light chain constant region;

[0435] The heavy chains A and B further comprise a first VHH and a second VHH that specifically bind CD16A, wherein the C-terminus of the first VHH and the second VHH are connected, via or without a connector, to the N-terminus of the Fab heavy chain variable region that specifically binds EGFR and the Fab heavy chain variable region that specifically binds CDH17; or the N-terminus of the first VHH and the second VHH are connected, via or without a connector, to the C-terminus of the first Fc region and the second Fc region; or

[0436] The light chains a and b further comprise a first VHH and a second VHH, respectively, wherein the C-terminus of the first VHH and the second VHH are connected, via or without a connector, to the N-terminus of the Fab light chain variable region that specifically binds EGFR and the Fab light chain variable region that specifically binds CDH17, respectively; or wherein the N-terminus of the first VHH and the second VHH are connected, via or without a connector, to the C-terminus of the Fab light chain variable region that specifically binds EGFR and the Fab light chain variable region that specifically binds CDH17.

[0437] The first VHH and the second VHH can be the same or different, preferably the same, for example, as defined in Section II-2 respectively;

[0438] Optionally, the heavy chain variable region and light chain variable region of the Fab that specifically binds to EGFR are as defined herein, for example as defined in Section II-3-1, or the Fab that specifically binds to EGFR is as defined in II-3-3;

[0439] Optionally, the heavy chain variable region and light chain variable region of a CDH17-specific Fab are as defined herein, for example as defined in Section II-1 or Section II-3-2, or the CDH17-specific Fab is as defined in II-3-3.

[0440] Optionally, the heavy chain constant region CH1 and the light chain constant region are as defined in Section II-3-3, respectively; and / or

[0441] Optionally, the first Fc region and the second Fc region are as defined in Section II-3-4.

[0442] In some implementations, the first Fc region contains S364R and D399K mutations, and the second Fc region contains Y349T, K370S, and K409D mutations; optionally, the first Fc region and the second Fc region also contain L234A / L235A mutations, respectively.

[0443] In some implementations, the light chain constant region in light chain a may be the same as or different from the light chain constant region in light chain b. For example, the light chain constant regions in light chains a and b are Kappa light chain constant regions.

[0444] In some specific embodiments, the trispecific antibody that specifically binds to EGFR, CD16A, and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0445] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0446] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0447] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0448] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0449] In some specific embodiments, the trispecific antibody that specifically binds to EGFR, CD16A, and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0450] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:75, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0451] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0452] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0453] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0454] In some specific embodiments, the trispecific antibody that specifically binds to EGFR, CD16A, and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0455] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0456] The light chain a comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0457] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0458] The light chain b comprises the amino acid sequence shown in SEQ ID NO:72, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0459] In some specific embodiments, the trispecific antibody that specifically binds to EGFR, CD16A, and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0460] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:77, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0461] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0462] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0463] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0464] In some specific embodiments, the trispecific antibody that specifically binds to EGFR, CD16A, and CDH17 comprises or is composed of heavy chain A, heavy chain B, light chain a, and light chain b, wherein...

[0465] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0466] The light chain a comprises the amino acid sequence shown in SEQ ID NO:79, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0467] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0468] The light chain b comprises the amino acid sequence shown in SEQ ID NO:73, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0469] In some embodiments, the trispecific antibody of the present invention comprises any one or more of the domains of P006, P002, P003, P004 and P005 shown in Example 9 (e.g., the domains shown as P006, P002, P003, P004 and P005 in Table 10), or all of the domains.

[0470] III. Polynucleotides, vectors, and host cells

[0471] The present invention provides nucleic acids encoding any of the antibody molecules of the present invention (e.g., the anti-CDH17 antibody of the present invention or its antigen-binding fragment, or the anti-CD16A-VHH antibody or its heavy chain antibody, or multispecific antibodies such as bispecific antibodies or trispecific antibodies).

[0472] In one aspect, the present invention provides a nucleic acid encoding any of the above, such as the anti-CDH17 antibody or its antigen-binding fragment, or the anti-CD16A-VHH antibody or its heavy chain antibody, or a multispecific antibody such as a bispecific antibody or a trispecific antibody.

[0473] To facilitate production and purification, for example, the anti-CDH17 antibody or its antigen-binding fragment, or the anti-CD16A-VHH antibody or its heavy chain antibody, or multispecific antibodies such as bispecific or trispecific antibodies of the present invention, may be fused with a secretory signal peptide at the N-terminus or C-terminus (e.g., C-terminus), and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label.

[0474] As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0475] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 6, 11, 15, 16, 20, 21, 25, 26, 34, 37, 38, 39, 40, 41, 42, 50, 59-68, 72-83, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 6, 11, 15, 16, 20, 21, 25, 26, 34, 37, 38, 39, 40, 41, 42, 50, 59-68, 72-83.

[0476] The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0477] In one aspect, the present invention also provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0478] In one aspect, the invention also provides a host cell comprising the nucleic acid or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, such as HEK 293 or 293F cells or 293FT cells or Expi293 cells). In yet another embodiment, the host cell is prokaryotic.

[0479] Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can also be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0. Suitable mammalian host cell lines for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO cell, a HEK293 cell, a 293FT cell, or an Expi293 cell. In some embodiments, the host cell is a CHO cell, such as a FUT8 knockout CHO cell.

[0480] IV. Production and purification of the molecules of this invention

[0481] In another aspect, the present invention provides a method for producing molecules of the present invention (e.g., antibody molecules of the present invention or antigen-binding fragments thereof, including multispecific antibodies such as bispecific or trispecific antibodies), the method comprising: culturing host cells containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing a polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chain to produce the molecule under conditions suitable for assembling the polypeptide chain into the molecule.

[0482] For recombinant production, a polynucleotide encoding the polypeptide chain of the molecule of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0483] The molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0484] The purity of the molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0485] V. Determination Method

[0486] The molecules of the present invention provided herein (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies)) can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. Examples illustrate methods for determining the properties of the anti-CDH17 antibody of the present invention or its antigen-binding fragment, or the anti-CD16A-VHH antibody or its heavy chain antibody, or multispecific antibodies such as bispecific or trispecific antibodies, such as thin-layer chromatography (BLI), ADCC killing effect detection, in vitro tumor proliferation inhibition experiments, in vitro safety studies, etc.

[0487] VI. Immunoconjugates

[0488] In one aspect, the present invention provides an immunoconjugate comprising the molecules of the present invention (e.g., the antibody molecules of the present invention or antigen-binding fragments thereof (including multispecific antibodies such as bispecific or trispecific antibodies) or immunoconjugates thereof) and one or more other active ingredients (e.g., active ingredients derived from medicaments or therapeutic agents for treating diseases of the present invention, such as small molecules or antitumor compounds, such as cytotoxic agents, that enhance the therapeutic effect of the molecules of the present invention).

[0489] In some implementations, the immune conjugate is an antibody-drug conjugate (ADC).

[0490] VII. Drugs and drug combinations

[0491] In some embodiments, the present invention also relates to compositions comprising the molecules of the present invention (e.g., antibody molecules of the present invention or antigen-binding fragments thereof, including multispecific antibodies such as bispecific or trispecific antibodies) or immunoconjugates thereof) (e.g., pharmaceutical compositions such as pharmaceutical formulations).

[0492] In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. In one embodiment, the composition, such as a pharmaceutical composition, comprises molecules of the present invention, and a combination of one or more other therapeutic agents.

[0493] The compositions of the present invention may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used herein, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, or absorption delay agents. For information on the use and applications of pharmaceutical excipients, see “Handbook of Pharmaceutical Excipients”, 8th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago. The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0494] A pharmaceutical product comprising the antibody molecule or immunoconjugate described herein can be prepared, for example, in the form of a lyophilized formulation or an aqueous solution, by mixing the antibody molecule of the present invention (e.g., the anti-CDH17 antibody of the present invention or its antigen-binding fragment or anti-CD16A-VHH antibody or its heavy chain antibody or multispecific antibody such as a bispecific antibody or a trispecific antibody) or immunoconjugate having the desired purity with one or more optional pharmaceutical excipients.

[0495] In some embodiments, the present invention also provides a pharmaceutical combination or combination of drugs comprising the antibody molecule or immunoconjugate of the present invention, and one or more other therapeutic agents, etc.

[0496] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0497] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0498] - A first container containing a pharmaceutical composition comprising the antibody molecule or immunoconjugate of the present invention;

[0499] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0500] In some embodiments, when the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies) or their immunoconjugates) are used to treat tumors, the therapeutic agents are, for example, various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0501] In a preferred embodiment, the other therapeutic agent is a small molecule drug.

[0502] VIII. Methods and Applications

[0503] This invention provides, in one aspect, a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., an antibody molecule of the invention or an antigen-binding fragment thereof (including multispecific antibodies such as bispecific or trispecific antibodies) or an immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a kit. In some embodiments, the disease is a CDH17-related disease and / or condition. In some embodiments, the disease is an EGFR-related disease and / or condition. In some embodiments, the disease is a CDH17 and EGFR-related disease and / or condition.

[0504] In some embodiments, the present invention relates to molecules of the invention (e.g., antibody molecules of the invention or antigen-binding fragments thereof, including multispecific antibodies such as bispecific or trispecific antibodies, or immunoconjugates thereof), pharmaceutical compositions, pharmaceutical combinations, or kits for activating the immune system.

[0505] In some embodiments, the present invention relates to molecules of the invention (e.g., antibody molecules of the invention or antigen-binding fragments thereof (including multispecific antibodies such as bispecific or trispecific antibodies) or immunoconjugates thereof), pharmaceutical compositions, pharmaceutical combinations or kits, for use in therapies, such as for treating CDH17-related diseases and / or conditions, EGFR-related diseases and / or conditions, or CDH17 and EGFR-related diseases and / or conditions.

[0506] In some embodiments, the present invention relates to methods of treating diseases, such as those mentioned herein, using molecules of the present invention (e.g., antibody molecules of the present invention or antigen-binding fragments thereof, including multispecific antibodies such as bispecific or trispecific antibodies) or immunoconjugates thereof), pharmaceutical compositions, pharmaceutical combinations or kits, or uses for the treatment described herein, or uses for the preparation of medicaments for the treatment described herein.

[0507] In some embodiments, the disease is a CDH17-related disease and / or condition, an EGFR-related disease and / or condition, or a CDH17 and EGFR-related disease and / or condition. In some embodiments, the disease is, for example, an oncology such as cancer.

[0508] In some embodiments, the tumor is a solid tumor or a hematologic malignancy (including lymphoma) and a metastatic lesion. In some embodiments, the tumor is a malignant tumor such as cancer. In one embodiment, examples of solid tumors include malignant solid tumors. The cancer can be in the early, intermediate, or late stage, or metastatic stage. In some embodiments, the tumor is a tumor immune evader.

[0509] In some embodiments, the tumor is a CDH17-positive tumor or cancer, or an EGFR-positive tumor or cancer, preferably a CDH17-positive tumor or cancer, or an EGFR and CDH17-positive tumor or cancer. In some embodiments, the cancer is colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer.

[0510] In some embodiments, a CDH17-positive tumor or cancer refers to abnormal expression (e.g., overexpression) or activity of CDH17 in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has CDH17 expression. In some embodiments, the subject (particularly an adult subject) has both CDH17 and EGFR expression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) CDH17 (optionally and EGFR) (e.g., compared to a healthy subject). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) CDH17 (optionally and EGFR) (e.g., compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject), or compared to CDH17 (optionally and EGFR) in adjacent healthy tissue or cells of the subject). In some embodiments, a CDH17-positive tumor or cancer refers to a tumor or cancer having tumor cells expressing CDH17 (optionally also expressing EGFR). In some embodiments, the tumor cells express CDH17, for example, moderately or highly expressing CDH17 (optionally, and EGFR). In some embodiments, a CDH17-positive tumor refers to the aberrant (e.g., overexpression) of CDH17 (optionally, and EGFR) in tumor cells. In some embodiments, aberrant CDH17 expression refers to higher CDH17 expression (optionally, higher EGFR expression) on tumor cells compared to control cells (e.g., healthy cells in a corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells).

[0511] In some embodiments, an EGFR and CDH17 positive tumor or cancer refers to abnormal expression (e.g., overexpression) or activity of EGFR and CDH17 in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has both CDH17 and EGFR expression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of EGFR and CDH17 (e.g., compared to healthy subjects). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of EGFR and CDH17 (e.g., compared to biological samples from healthy subjects (e.g., corresponding tissues or cells in healthy subjects), or compared to EGFR and CDH17 in adjacent healthy tissues or cells of the subject). In some embodiments, an EGFR and CDH17 positive tumor or cancer refers to said tumor or cancer having tumor cells expressing EGFR and CDH17. In some embodiments, the individual's tumor cells express EGFR and CDH17, for example, moderate or high expression of EGFR and CDH17. In some implementations, EGFR and CDH17 positive tumors refer to tumor cells that abnormally express (e.g., overexpress) EGFR and CDH17. In some implementations, abnormal expression of EGFR and CDH17 refers to higher expression of EGFR and CDH17 on tumor cells compared to control cells (e.g., healthy cells in the corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells).

[0512] In some embodiments, an EGFR-positive tumor or cancer refers to abnormal expression (e.g., overexpression) or activity of EGFR in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has EGFR expression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of EGFR (e.g., compared to healthy subjects). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of EGFR (e.g., compared to biological samples from healthy subjects (e.g., corresponding tissues or cells in healthy subjects), or compared to EGFR in adjacent healthy tissues or cells of the subject). In some embodiments, an EGFR-positive tumor or cancer refers to said tumor or cancer having tumor cells that express EGFR. In some embodiments, the individual's tumor cells express EGFR, e.g., moderate or high expression of EGFR. In some embodiments, an EGFR-positive tumor refers to abnormal expression (e.g., overexpression) of EGFR in tumor cells. In some implementations, aberrant EGFR expression refers to higher EGFR expression in tumor cells compared to control cells (e.g., healthy cells in the corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells).

[0513] In some embodiments, the tumors or cancers suitable for prevention or treatment using the antibody molecules of the present invention are CDH17-positive tumors or cancers, CDH17 and EGFR-positive tumors or cancers, or EGFR-positive tumors or cancers. In a preferred embodiment, the EGFR and CDH17-positive tumors or cancers refer to tumors or cancers exhibiting high EGFR expression and high, intermediate, or low CDH17 expression in tumor cells.

[0514] In one specific embodiment, the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies) or their immunoconjugates) are capable of killing tumor cells and / or inhibiting tumor cell proliferation, such as tumor cells expressing EGFR or CDH17, preferably tumor cells expressing EGFR and CDH17, more preferably tumor cells that highly express EGFR and highly, moderately or poorly express CDH17, and / or tumors or cancers having KRAS wild-type overexpression or KRAS G12 mutation.

[0515] In one specific implementation, the molecules of the present invention can be used for anti-tumor effects.

[0516] In some embodiments, the tumor or cancer suitable for application in this invention is a KRAS G12-mutated tumor or cancer, for example, wherein (e.g., in the tumor tissue or cells) KRAS with a mutation at G12 is expressed, such as a tumor or cancer expressing KRAS with G12D, G12C, or G12V mutations. In some embodiments, the tumor or cancer suitable for application in this invention is colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer, such as colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer with KRAS wild-type overexpression or KRAS mutations, such as colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer with KRAS mutations of G12D, G12C, or G12V. In some embodiments, the tumor exhibits tumor immune evasion.

[0517] In some embodiments, the tumors or cancers suitable for use in this invention are CDH17 positive or CDH17 and EGFR positive, and are KRAS wild-type overexpressing or KRAS G12 mutated tumors or cancers, for example, wherein (e.g., said tumor tissue or cells) KRAS with a mutation at G12 is expressed, such as tumors or cancers expressing KRAS with G12D, G12C or G12V mutations.

[0518] In some embodiments, the tumor or cancer suitable for application in this invention is colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer, such as CDH17 positive or CDH17 and EGFR positive, and having KRAS wild-type overexpression or KRAS mutations, such as colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer with KRAS mutations of G12D, G12C, or G12V.

[0519] In some embodiments, the tumor is a tumor that has already been treated with other treatments such as chemotherapy and / or radiotherapy.

[0520] Depending on their therapeutic use, the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies) or their immunoconjugates) or pharmaceutical compositions may also be administered in combination with one or more other therapies, such as other modes of treatment and / or other therapeutic agents (i.e., for combination therapy), for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0521] In some implementation schemes, treatment methods include surgery; radiotherapy; local irradiation; or focused irradiation.

[0522] In other respects, the present invention provides the use of the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies) or their immunoconjugates) or compositions or pharmaceutical combinations comprising the present invention in the manufacture or preparation of a medicament for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0523] In other respects, the present invention also provides molecules of the present invention (e.g., antibody molecules of the present invention or antigen-binding fragments thereof (including multispecific antibodies such as bispecific or trispecific antibodies) or immunoconjugates thereof), or compositions or pharmaceutical combinations comprising thereof, for use in therapies, such as for treating the related diseases or conditions mentioned herein.

[0524] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0525] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the administration of the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies) or their immunoconjugates) or compositions containing them, such as pharmaceutical compositions, may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceutical agents.

[0526] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0527] The composition can also be applied topically via an implantable membrane, a sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In some embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecules via diffusion, timed release of a large pellet, or continuous administration.

[0528] IX. Other therapeutic agents

[0529] In some embodiments, other therapeutic agents that may be combined or administered in combination with the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments (including multispecific antibodies such as bispecific or trispecific antibodies) or their immunoconjugates) cover a wide range of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0530] In some embodiments, the other therapeutic agents are small molecule drugs, such as those selected from KRAS small molecule inhibitors, such as KARS-G12C inhibitors like fuzelase; or KRAS-G12D inhibitors like MRTX1133; or VEGF small molecule inhibitors like fruquintinib.

[0531] X Specific Implementation Plan

[0532] 1. An anti-CDH17 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising...

[0533] The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:21, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:16; or

[0534] The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:26.

[0535] 2. An anti-CDH17 antibody or its antigen-binding fragment thereof, said antibody or its antigen-binding fragment comprising a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein...

[0536] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23 or 27, SEQ ID NO:24, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or respectively composed of the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23 or 27, SEQ ID NO:24, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19.

[0537] 3. The anti-CDH17 antibody or its antigen-binding fragment according to embodiment 1 or 2, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), wherein the heavy chain variable region

[0538] a) comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:21; or comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:21; or

[0539] b) Containing, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:25; or containing, or consisting of, the amino acid sequence shown in SEQ ID NO:25.

[0540] 4. The anti-CDH17 antibody or antigen-binding fragment thereof according to any one of embodiments 1-3, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region

[0541] a) comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16; or comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 16; or

[0542] b) Contains, or consists of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26; or contains, or consists of, the amino acid sequence shown in SEQ ID NO:26.

[0543] 5. An anti-CDH17 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0544] a) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; or

[0545] b) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VH composed of said amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VH composed of said amino acid sequence; or

[0546] c) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:16; or

[0547] d) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:26.

[0548] 6. The anti-CDH17 antibody or its antigen-binding fragment as described in embodiment 1 or 2, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein

[0549] a) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; or

[0550] b) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or

[0551] c) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:16; or

[0552] d) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:26.

[0553] 7. The anti-CDH17 antibody or its antigen-binding fragment according to any one of embodiments 1-6, further comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, for example, the heavy chain constant region.

[0554] a) Contains or consists of an amino acid sequence selected from SEQ ID NO:55; or

[0555] b) Containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:55; and / or

[0556] The light chain constant region is a lambda or Kappa light chain constant region, for example, the Kappa light chain constant region, such as the human lambda or Kappa light chain constant region, for example, the light chain constant region.

[0557] a) Containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 43 or 56; or

[0558] b) Contains or consists of an amino acid sequence of SEQ ID NO:43 or 56.

[0559] 8. The anti-CDH17 antibody or its antigen-binding fragment as described in any one of embodiments 1-7, wherein the antibody is a monoclonal antibody.

[0560] 9. The anti-CDH17 antibody or its antigen-binding fragment as described in any one of embodiments 1-8, wherein the antibody is a humanized antibody or a chimeric antibody.

[0561] 10. The anti-CDH17 antibody or its antigen-binding fragment as described in any one of embodiments 1-9, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), bivalent antibody, or linear antibody.

[0562] 11. The anti-CDH17 antibody or antigen-binding fragment thereof as described in any one of embodiments 1-10, wherein the anti-CDH17 antibody is a bispecific antibody or a multispecific antibody, comprising a first binding specificity against CDH17 and a binding specificity against one or more other antigens.

[0563] 12. The anti-CDH17 antibody or its antigen-binding fragment as described in Implementation Scheme 11, wherein the other antigen is EGFR; or the other antigen is EGFR and CD16A.

[0564] 13. A multispecific antibody comprising a first antigen-binding region that specifically binds to EGFR, a second antigen-binding region that specifically binds to CDH17, and optionally a third antigen-binding region that specifically binds to CD16A.

[0565] 14. The multispecific antibody of implementation scheme 13, which is a bispecific antibody, and which includes a first antigen-binding region that specifically binds to EGFR and a second antigen-binding region that specifically binds to CDH17.

[0566] 15. The multispecific antibody of implementation scheme 13, which is a trispecific antibody, and which includes a first antigen-binding region that specifically binds to EGFR, a second antigen-binding region that specifically binds to CDH17, and a third antigen-binding region that specifically binds to CD16A.

[0567] 16. The multispecific antibody according to any one of embodiments 13-15, wherein the second antigen-binding region specifically binds to CDH17 and comprises VH and VL, wherein

[0568] a) The VH includes HCDR1, HCDR2, and HCDR3 as defined in embodiment 1 or 2, and the VL includes LCDR1, LCDR2, and LCDR3 as defined in embodiment 1 or 2; or

[0569] b) The VH comprises three complement-determining regions (HCDRs) of heavy chain variable regions, HCDR1, HCDR2 and HCDR3, and the VL comprises three complement-determining regions (LCDRs) of light chain variable regions, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:97; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:98; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:99; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:100; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:101; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:102.

[0570] 17. The multispecific antibody according to any one of embodiments 13-15, wherein the second antigen-binding region specifically binds to CDH17 and comprises VH and VL, wherein

[0571] a) The VH and the VL are VH and VL as defined in any one of embodiments 3-6; or b) The VH contains or consists of the amino acid sequence shown in SEQ ID NO:88, and the VL contains or consists of the amino acid sequence shown in SEQ ID NO:89.

[0572] 18. The multispecific antibody according to any one of embodiments 13-17, wherein the first antigen-binding region comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 are HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and LCDR1, LCDR2 and LCDR3 are LCDR1, LCDR2 and LCDR3 of the light chain variable region as shown in SEQ ID NO:2.

[0573] 19. The multispecific antibody according to any one of embodiments 13-18, wherein the first antigen-binding region comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:8; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:9; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:10; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:3; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:4; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:5.

[0574] 20. The multispecific antibody according to any one of embodiments 13-19, wherein the first antigen-binding region comprises VH and VL, wherein VH comprises or is composed of the sequence shown in SEQ ID NO:7; and VL comprises or is composed of the sequence shown in SEQ ID NO:2.

[0575] 21. The multispecific antibody according to any one of embodiments 13-20, wherein the second antigen-binding region is a Fab that specifically binds to CDH17; and / or the first antigen-binding region is a Fab that specifically binds to EGFR.

[0576] 22. The multispecific antibody according to embodiment 21, wherein the Fab serving as the first antigen-binding region or the second antigen-binding region comprises CH1, wherein CH1 is CH1 derived from IgG1, IgG2, IgG3, or IgG4, preferably CH1 derived from IgG1, for example, CH1

[0577] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:44; or

[0578] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:44.

[0579] 23. The multispecific antibody according to embodiment 21 or 22, wherein the Fab serving as the first antigen-binding region or the second antigen-binding region comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, for example, the Kappa light chain constant region.

[0580] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:43; or

[0581] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:43; or

[0582] The Lambda light chain constant region

[0583] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:56; or

[0584] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:56.

[0585] 24. The multispecific antibody according to any one of embodiments 21-23, wherein the Fab serving as the first antigen-binding region comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises

[0586] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49;

[0587] (ii) Contains or is composed of the amino acid sequence of SEQ ID NO:49; and

[0588] The Fab light chain includes

[0589] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1;

[0590] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:1.

[0591] 25. The multispecific antibody according to any one of embodiments 21-24, wherein the second antigen-binding region is the Fab of the antibody as defined in any one of embodiments 1-10.

[0592] 26. The multispecific antibody according to any one of embodiments 21-24, wherein the Fab serving as the second antigen-binding region comprises a Fab heavy chain and a Fab light chain, wherein

[0593] The Fab heavy chain includes

[0594] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:50;

[0595] (ii) Contains or is composed of the amino acid sequence of SEQ ID NO:50; and

[0596] The Fab light chain includes

[0597] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:15;

[0598] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:15; or

[0599] The Fab heavy chain includes

[0600] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 90;

[0601] (ii) Contains or is composed of the amino acid sequence of SEQ ID NO:90; and

[0602] The Fab light chain includes

[0603] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:92;

[0604] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:92.

[0605] 27. The multispecific antibody according to any one of embodiments 13-26, wherein the multispecific antibody is an IgG-like multispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different, optionally, the Fc regions are human IgG1, IgG2, IgG3 or IgG4 Fc, optionally, the Fc regions contain

[0606] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 57 or 58; or

[0607] (ii) Contains or consists of an amino acid sequence of SEQ ID NO:57 or 58.

[0608] 28. The multispecific antibody according to embodiment 27, wherein the two Fc regions are different, wherein mutations are introduced into the first Fc region and the second Fc region based on Innobody technology to promote heterodimerization of the first Fc region and the second Fc region.

[0609] 29. The multispecific antibody of implementation scheme 28, wherein the CH3 of one Fc region contains S364R and D399K mutations, and the CH3 mutation of the other Fc region contains Y349T, K370S and K409D mutations.

[0610] 30. The multispecific antibody according to any one of embodiments 27-29, wherein the first and / or second Fc region contains the L234A / L235A mutation.

[0611] 31. The multispecific antibody according to any one of implementation schemes 27-30, wherein

[0612] a) One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:47, and the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:48;

[0613] b) One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:86, and the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:87;

[0614] c) One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:86 and contains the mutations S364R and D399K, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87 and contains the mutations Y349T, K370S, and K409D; or

[0615] d) One Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47 and contains the mutations S364R and D399K and L234A / L235A mutations, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:48 and contains the mutations Y349T, K370S, and K409D and L234A / L235A mutations.

[0616] 32. The multispecific antibody according to any one of embodiments 13-31, wherein the third antigen-binding region is a VHH that specifically binds to CD16A.

[0617] 33. The multispecific antibody of embodiment 32, wherein the VHH antibody that specifically binds to CD16A comprises the three complementarity-determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40 or 41.

[0618] 34. The multispecific antibody according to embodiment 32 or 33, wherein the VHH that specifically binds to CD16A comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein

[0619] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:12, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:14, 36, or 42.

[0620] (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:29 or 33, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31; or

[0621] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:36 or 14.

[0622] 35. The multispecific antibody according to any one of embodiments 32-34, wherein the VHH that specifically binds to CD16A comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region

[0623] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40, or 41; or

[0624] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40 or 41.

[0625] 36. The multispecific antibody according to any one of embodiments 13-35, wherein it is a bispecific antibody and comprises or consists of the following chains:

[0626] Heavy chain A: from the N end to the C end, it contains or consists of the following: first Fab heavy chain variable region - heavy chain constant region CH1 - first Fc region;

[0627] Light chain a: from the N end to the C end, it includes or consists of the following: the light chain variable region of the first Fab - the light chain constant region;

[0628] Heavy chain B: From the N end to the C end, it contains or consists of the following: second Fab heavy chain variable region - heavy chain constant region CH1 - second Fc region;

[0629] Light chain b: From the N end to the C end, it includes the following components: the light chain variable region of the second Fab - the light chain constant region;

[0630] The first antigen-binding region is a first Fab that specifically binds to EGFR, and the second antigen-binding region is a second Fab that specifically binds to CDH17; the heavy chain variable region and the light chain variable region of the first Fab are as defined in any one of embodiments 18-20; and the heavy chain variable region and the light chain variable region of the second Fab are as defined in embodiment 16 or 17; or the heavy chain and light chain of the first Fab are as defined in embodiment 24; and the heavy chain and light chain of the second Fab are as defined in embodiment 25 or 26.

[0631] The heavy chain constant region CH1 is as defined in embodiment 22 and the light chain constant region is as defined in embodiment 23; and

[0632] The first Fc region and the second Fc region are as defined in any of embodiments 27-31.

[0633] 37. The multispecific antibody described in Implementation Scheme 36, wherein...

[0634] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:70, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0635] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0636] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0637] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0638] or

[0639] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:70, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0640] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0641] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:91, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0642] The light chain b comprises the amino acid sequence shown in SEQ ID NO:92, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0643] or

[0644] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:74, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0645] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0646] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0647] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0648] 38. The multispecific antibody according to any one of embodiments 13 and 15-35, which is a trispecific antibody, comprising a first antigen-binding region first Fab that specifically binds to EGFR, a second antigen-binding region second Fab that specifically binds to CDH17, and a third antigen-binding region first VHH and second VHH that specifically binds to CD16A, and optionally comprising a first Fc region and a second Fc region.

[0649] The heavy chain variable region and the light chain variable region of the first Fab are defined as in any one of embodiments 18-20; and the heavy chain variable region and the light chain variable region of the second Fab are defined as in embodiment 16 or 17; or the heavy chain and light chain of the first Fab are defined as in embodiment 24; and the heavy chain and light chain of the second Fab are defined as in embodiment 25 or 26.

[0650] The first VHH and the second VHH are respectively defined as in any one of embodiments 33-35; and

[0651] The first Fc region and the second Fc region are as defined in any of embodiments 27-31.

[0652] 39. The multispecific antibody according to embodiment 38, wherein the first VHH and the second VHH are respectively linked to the N-terminus of the Fab heavy chain of the first Fab and the second Fab, or respectively inserted between the C-terminus and the Fc region of the Fab heavy chain of the first Fab and the second Fab, or respectively linked to the N-terminus or C-terminus of the Fab light chain of the first Fab and the second Fab, or respectively linked to the C-terminus of the first Fc region and the second Fc region; optionally, the first VHH and the second VHH are the same or different.

[0653] 40. The multispecific antibody according to embodiment 39, comprising or consisting of the following:

[0654] Heavy chain A: from the N end to the C end, it contains or consists of the following: first Fab heavy chain variable region - heavy chain constant region CH1 - first Fc region;

[0655] Light chain a: from the N end to the C end, it includes or consists of the following: the light chain variable region of the first Fab - the light chain constant region;

[0656] Heavy chain B: From the N end to the C end, it contains or consists of the following: second Fab heavy chain variable region - heavy chain constant region CH1 - second Fc region;

[0657] Light chain b: From the N end to the C end, it includes the following components: the light chain variable region of the second Fab - the light chain constant region;

[0658] The first Fab specifically binds to the first antigen, and the second Fab specifically binds to the second antigen;

[0659] The heavy chains A and B further comprise a first VHH and a second VHH, respectively, wherein the C-ends of the first VHH and the second VHH are connected, with or without a connector, to the N-ends of the first Fab heavy chain variable region and the second Fab heavy chain variable region; or the N-ends of the first VHH and the second VHH are connected, with or without a connector, to the C-ends of the first Fc region and the second Fc region; or

[0660] The light chains a and b further include a first VHH and a second VHH, wherein the C-ends of the first VHH and the second VHH are connected to the N-ends of the first Fab light chain variable region and the second Fab light chain variable region via or without a connector, or wherein the N-ends of the first VHH and the second VHH are connected to the C-ends of the first Fab light chain variable region and the second Fab light chain variable region via or without a connector.

[0661] The heavy chain variable region and the light chain variable region of the first Fab are defined as in any one of embodiments 18-20; and the heavy chain variable region and the light chain variable region of the second Fab are defined as in embodiment 16 or 17; or the heavy chain and light chain of the first Fab are defined as in embodiment 24; and the heavy chain and light chain of the second Fab are defined as in embodiment 25 or 26.

[0662] The first VHH and the second VHH are respectively defined as in any one of embodiments 33-35;

[0663] The heavy chain constant region CH1 is as defined in embodiment 22 and the light chain constant region is as defined in embodiment 23; and

[0664] The first Fc region and the second Fc region are as defined in any of embodiments 27-31.

[0665] 41. The multispecific antibody of embodiment 40, wherein the first VHH and the second VHH are respectively linked via a linker, the linker comprising (G) n , where n = any integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5 or 10, for example, the connector contains or is composed of the amino acid sequence shown in SEQ ID NO: 45 or 46.

[0666] 42. The multispecific antibody according to embodiment 40 or 41, wherein

[0667] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:75, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0668] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0669] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:81, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0670] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0671] or

[0672] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0673] The light chain a comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0674] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0675] The light chain b comprises the amino acid sequence shown in SEQ ID NO:72, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0676] or

[0677] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:77, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0678] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0679] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:83, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0680] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0681] or

[0682] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0683] The light chain a comprises the amino acid sequence shown in SEQ ID NO:79, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0684] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0685] The light chain b comprises the amino acid sequence shown in SEQ ID NO:73, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0686] 43. The multispecific antibody according to embodiment 39, comprising or consisting of the following:

[0687] Heavy chain A: From the N end to the C end, it includes or consists of the following: first Fab heavy chain variable region - heavy chain constant region CH1 - first VHH - first Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first VHH and the first VHH is connected at its C end to the N end of the first Fc region via or without a connector.

[0688] Light chain a: from the N end to the C end, it includes or consists of the following: the light chain variable region of the first Fab - the light chain constant region;

[0689] Heavy chain B: From the N end to the C end, it includes or consists of the following: second Fab heavy chain variable region - heavy chain constant region CH1 - second VHH - second Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the second VHH and the second VHH is connected at its C end to the N end of the second Fc region via or without a connector.

[0690] Light chain b: From the N end to the C end, it includes the following components: the light chain variable region of the second Fab - the light chain constant region;

[0691] The heavy chain variable region and the light chain variable region of the first Fab are defined as in any one of embodiments 18-20; and the heavy chain variable region and the light chain variable region of the second Fab are defined as in embodiment 16 or 17; or the heavy chain and light chain of the first Fab are defined as in embodiment 24; and the heavy chain and light chain of the second Fab are defined as in embodiment 25 or 26.

[0692] The first VHH and the second VHH are respectively defined as in any one of embodiments 33-35;

[0693] The heavy chain constant region CH1 is as defined in embodiment 22 and the light chain constant region is as defined in embodiment 23; and

[0694] The first Fc region and the second Fc region are as defined in any of embodiments 27-31.

[0695] 44. The multispecific antibody according to embodiment 43, wherein the first or second VHH is connected to the C-terminus of the first or second Fab heavy chain at its N-terminus via a linker 1, and the first or second VHH is connected to the N-terminus of the first or second Fc region at its C-terminus via a linker 2.

[0696] 45. The multispecific antibody according to embodiment 44, wherein the linker 1 or 2 comprises (G) n , where n = any integer from 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5 or 10, for example, connector 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:45, and / or connector 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:46.

[0697] 46. ​​The multispecific antibody according to any one of embodiments 43-45, comprising or composed of heavy chain A, heavy chain B, light chain a and light chain b, wherein...

[0698] Heavy chain A comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0699] The light chain a comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0700] Heavy chain B comprises the amino acid sequence shown in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0701] The light chain b comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0702] 47. VHH antibody that specifically binds to CD16A, which contains

[0703] The three complementary determining regions (CDRs) contained in the VHH shown in SEQ ID NO: 11, 32, 34, 37, 38, 39, 40 or 41.

[0704] 48. The VHH antibody that specifically binds to CD16A according to implementation plan 47, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein...

[0705] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:12, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:14, 36, or 42.

[0706] (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:33, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31; or

[0707] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:36 or 14.

[0708] 49. The VHH antibody that specifically binds to CD16A according to embodiment 47 or 48, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0709] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11, 32, 34, 37, 38, 39, 40, or 41; or

[0710] (ii) Contains or consists of an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO: 11, 32, 34, 37, 38, 39, 40 or 41.

[0711] 50. A heavy chain antibody that specifically binds to CD16A, comprising a VHH antibody that specifically binds to CD16A as described in any one of embodiments 47-49.

[0712] 51. The CD16A-specific heavy chain antibody of embodiment 50, comprising a heavy chain, said heavy chain comprising or consisting of any one of embodiments 47-49 of the VHH antibody specifically binding to CD16A linked to a constant region or Fc region of the antibody heavy chain.

[0713] 52. The heavy chain antibody that specifically binds to CD16A according to embodiment 51, wherein the constant region of the antibody heavy chain is as defined in embodiment 7, or the Fc region is as defined in embodiment 27, optionally the Fc region further comprising an L234A / L235A mutation.

[0714] 53. The heavy chain antibody of embodiment 52 that specifically binds to CD16A, comprising or consisting of a heavy chain, wherein the heavy chain

[0715] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 60-67; or

[0716] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:60-67.

[0717] 54. A heavy chain antibody that specifically binds to CD16A, as described in any of embodiments 51-53, comprising two dimerized heavy chains, wherein the two heavy chains may be identical or different.

[0718] 55. A VHH antibody that specifically binds to CD16A from any of embodiments 46-49, or a heavy chain antibody that specifically binds to CD16A from any of embodiments 50-54, wherein the antibody is a chimeric antibody or a humanized antibody.

[0719] 56. A nucleic acid molecule comprising any one chain encoding an antibody or antigen-binding fragment thereof of any one of embodiments 1-12, or a multispecific antibody of any one of embodiments 13-46, or a VHH antibody specifically binding to CD16A of any one of embodiments 47-49 and 55, or a heavy chain antibody specifically binding to CD16A of any one of embodiments 50-55, or composed of said polynucleotide.

[0720] 57. An expression vector comprising the nucleic acid molecule of embodiment 56, preferably, said expression vector is pCDNA, such as pCDNA3.1.

[0721] 58. A host cell comprising the nucleic acid molecule of embodiment 56 or the expression vector of embodiment 57, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293F cells.

[0722] 59. A method for preparing the antibody or antigen-binding fragment thereof of any one of embodiments 1-12, or the multispecific antibody of any one of embodiments 13-46, or the VHH antibody specifically binding to CD16A of any one of embodiments 47-49 and 55, or the heavy chain antibody specifically binding to CD16A of any one of embodiments 50-55, the method comprising culturing a host cell of the nucleic acid molecule of embodiment 56 or the expression vector of embodiment 57 under conditions suitable for expression of the polypeptide chain of the antibody or the antigen-binding fragment thereof, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0723] 60. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-12 or the multispecific antibody of any one of embodiments 13-46, for example, an antibody-drug conjugate.

[0724] 61. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-12, or the multispecific antibody of any one of embodiments 13-46, or the VHH antibody specifically binding to CD16A of any one of embodiments 47-49 and 55, or the heavy chain antibody specifically binding to CD16A of any one of embodiments 50-55, or the immunoconjugate of embodiment 60, and optionally a pharmaceutical excipient.

[0725] 62. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-12, or the multispecific antibody of any one of embodiments 13-46, or the VHH antibody specifically binding to CD16A of any one of embodiments 47-49 and 55, or the heavy chain antibody specifically binding to CD16A of any one of embodiments 50-55, or the immunoconjugate of embodiment 60, and one or more other therapeutic agents, such as said therapeutic agents being various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). Preferably, said small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS-G12C inhibitors, such as fuzelase; or KRAS-G12D inhibitors, such as MRTX1133; or VEGF small molecule inhibitors, such as fruquintinib.

[0726] 63. A method for preventing or treating tumors in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-12, or a multispecific antibody of any one of embodiments 13-46, or a VHH antibody specifically binding to CD16A of any one of embodiments 47-49 and 55, or a heavy chain antibody specifically binding to CD16A of any one of embodiments 50-55, or an immunoconjugate of embodiment 60, or a pharmaceutical composition or formulation of embodiment 61, or a pharmaceutical combination of embodiment 62.

[0727] 64. The method of embodiment 63, wherein the tumor is a solid tumor or hematologic malignancy (including lymphoma), such as a CDH17-positive tumor or cancer, or a CDH17 and EGFR-positive tumor or cancer, such as colorectal cancer, colon cancer (e.g., colon adenocarcinoma), or rectal cancer, such as a KRAS-mutated tumor or cancer, such as a tumor or cancer with a KRAS G12 mutation (e.g., G12D, G12C, or G12V mutation).

[0728] 65. The method of embodiment 63 or 64, wherein the method further comprises administration in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents, preferably, the therapeutic agents being various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immune checkpoint inhibitors or agonists), preferably, the small molecule drug being selected from KRAS small molecule inhibitors, such as KRAS-G12C inhibitors, such as fuzelase; or KRAS-G12D inhibitors, such as MRTX1133; or VEGF small molecule inhibitors, such as fruquintinib.

[0729] Example

[0730] Example 1. Preparation of anti-CDH17 antibody from hybridoma

[0731] The full-length GS-huCDH17 cell line was constructed using the following method:

[0732] 1. The plasmid encoding the nucleic acid of PvuI (NEB) and 10×cut smart buffer, CDH17-FL (SEQ ID NO:51) was transfected into pcDNA3.1 plasmid and reacted overnight at 37°C to digest the plasmid with enzymes.

[0733] 2. 24 hours before transfection, administer 0.6×10 6 GS-CHO cells were passaged at a cell density of cells / ml and cultured on a shaker at 37°C, 6% CO2, and 120 rpm.

[0734] 3. Take 1.2 × 10 7 Transfer the cells to a 50ml centrifuge tube, centrifuge at 200g for 5 minutes, and discard the supernatant. Wash twice.

[0735] 4. Add the enzyme-digested plasmid into the cells and perform electroporation. After electroporation, immediately transfer the cells to preheated fresh culture medium and incubate on a shaker at 37°C, 6% CO2, and 120 rpm.

[0736] 5. After 16 hours of electroporation, centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Add 1% SP4 (Lonza) and 50 μM L-methionine sulfonamide MSX (SIGMA) to the culture medium to adjust the cell density to 5E. 5 / mL, incubated under pressure.

[0737] Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized. The GS-huCDH17 full-length cell line constructed above was injected intraperitoneally into the mice, and immunization was performed once every week for a total of 3 times. One week later, the extracellular Domain1-2 protein (SEQ ID NO:52) was emulsified with Freund's adjuvant (Freund's complete adjuvant CFA: sigma, #SLBV0593; Freund's incomplete adjuvant IFA: sigma, #SLBT6189) and administered twice more, with subcutaneous injection of 25 μg protein per mouse every two weeks. One week after the completion of immunization, serum titers were measured.

[0738] Once the serum titer met the requirements, the spleen of the mouse was harvested to prepare a suspension of B lymphocytes, which were then electrofused with SP2 / 0 myeloma cells (ATCC, #CRL-1581). The fused cells were diluted to 1–2 × 10⁴ cells / ml with selective medium (1640 medium containing 20% ​​FBS and 1x HAT), and seeded into 96-well plates, with 100 μl of cell suspension added to each well. On day 7 post-fusion, the selection medium (1640 medium containing 10% FBS and 1x HT) was replaced. The supernatant was collected for analysis after day 10 (or longer, depending on cell growth status).

[0739] Hybridoma clones that bound to both human and monkey CDH17-overexpressing CHO cells were screened using flow cytometry (FACS). Positive clones were further subjected to ELISA and affinity assays. Finally, 25 clones were selected for subcloning using limiting dilution, and single-clone cells were picked. The supernatant from the single-clone cell wells was used to repeat the screening process, selecting target positive wells, expanding the culture, and then cryopreserving the cells.

[0740] Example 2. Preparation of CDH17 chimeric antibody

[0741] The antibody light and heavy chain gene sequences of the 25 hybridoma candidate clones obtained in Example 1 were retrieved, and clones were selected based on sequence similarity to construct human-mouse chimeric antibodies.

[0742] Approximately 5 × 10⁶ freshly cultured cells were collected, and RNA was extracted (Macherey-Nagel, Cat#740984.250). The light and heavy chain variable region gene fragments of correctly paired clones were then processed using homologous recombinase (…). (Catalog number: C112-01) is ligated into the pcDNA3.1 vector (containing the heavy chain constant region SEQ ID NO:55 or the light chain constant region SEQ ID NO:43) to obtain expression plasmids for light chain and heavy chain antibodies.

[0743] The light chain plasmid and heavy chain plasmid of the same antibody were mixed at a 1:1 molar ratio and transfected into 293F cells with polyethyleneimine (PEI) (Polysciences, Cat#23966). After culturing for 5-7 days, when the cell viability was less than 60%, the cell culture supernatant was collected and purified into monoclonal antibody using a Protein A affinity column.

[0744] Table 1: VH and VL mouse anti-sequences of 68A7E3

[0745] Example 3. CDH17 chimeric antibody affinity assay

[0746] The equilibrium dissociation constants (KD) of the antibodies of this invention binding to different domains of human extracellular proteins (full length human CDH17, FL, SEQ ID NO:51, human CDH17 (Domain 1–Domain 2, D1-D2, SEQ ID NO:52), and human CDH17 (Domain 5–Domain 7, D5-D7, SEQ ID NO:54)) were determined using ForteBio thin-layer interferometry. ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).

[0747] In short, the AHQ (Pall, 1506091) sensor was equilibrated offline in the analysis buffer for 30 minutes, followed by online detection for 60 seconds to establish a baseline. The purified antibody obtained as described above was then loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to the antigen CDH17, and subsequently transferred to the analysis buffer for dissociation rate measurement. KD values ​​were analyzed using ForteBio analysis software.

[0748] The results of the detection of the affinity of the finally selected molecule 68A7E3 and the binding domain of CDH17 are shown in Table 2. The affinity of 68A7E3 is 1.94E-09, and it binds to the D1-D2 domain of human CDH17.

[0749] Table 2: Results of chimeric antibody affinity and CDH17 binding domain detection

[0750] Example 4. Humanization of CDH17 antibody

[0751] The amino acid sequences of the heavy chain variable region and light chain variable region of one of the antibodies (68A7E3) obtained in this invention are shown in Table 1.

[0752] The obtained antibody sequence was then humanized through the following steps:

[0753] 1. Determine the CDR ring structure of VH and VL respectively, see Appendix Table 3;

[0754] 2. Find the closest homologous sequences for regions V and J respectively in the phylogenetic sequence database;

[0755] 3. Graft the CDR region of the antibody onto the human framework region;

[0756] 4. Analyze sequence and structural features to determine the amino acid positions in the framework region that maintain CDR function;

[0757] 5. Analyze whether reverse mutations have been performed at important amino acid positions;

[0758] 6. Optimize amino acids at risk sites.

[0759] Please refer to the attached sequence listing for the variable region amino acid sequence of the humanized antibody obtained in this invention.

[0760] Table 3: CDH17 antibody sequence

[0761] Table 4: Amino acid sequence of CDR of CDH17 antibody

[0762] Antibodies 68A7E3 and Hz68A7E3 were prepared as described in Example 2, and their affinity for the full-length human CDH17 protein was measured as described in Example 3 (Table 5). The binding of antibodies 68A7E3 and Hz68A7E3 to C99 cells (Nanjing Kebai, CBP60833) was also measured, and the results are shown in Figure 16.

[0763] Table 5: Affinity of Humanized Antibodies

[0764] Cell binding detection method:

[0765] 1. Cell Plating. Digest cells with EDTA (Gibco, 15040-066). After cell detachment, stop the process with culture medium. Collect cells into centrifuge tubes, centrifuge at 400g for 4 min, resuspend in culture medium, and plate into 96-well round-bottom plates (COSTAR, 3799), 2 x 10⁶ cells / well.5 cells / well.

[0766] Centrifuge at 2,500 g for 4 min, and wash once with FACS Buffer (2% FBS in PBS). Prepare a live / dead dye (Invitrogen, L34976) using FACS Buffer:live / dead dye = 1000. Resuspend cells at 100 μl / well and incubate at room temperature for 20 min.

[0767] 3. After incubation, wash twice with FACS Buffer and resuspend cells in 50 μl / well of FACS Buffer. Prepare 12 serial dilutions of the test antibody 68A7E3 and Hz68A7E3 at a final concentration of 300 nM, using a 4-fold serial dilution. Add 50 μl / well of each antibody to the cells, mix well, and incubate at 4°C for 1 h.

[0768] 4. After incubation, wash twice with FACS Buffer, prepare secondary antibody (Biolegend, 366904) with FACS Buffer: secondary antibody = 100, resuspend cells at 100 μl / well, and incubate at 4°C in the dark for 1 h.

[0769] 5. After incubation, wash twice with FACS Buffer, resuspend cells in 100 μl / well FACS Buffer, and perform flow cytometry analysis (BD, FACSymphony).

[0770] Figure 16 shows that the humanized antibody Hz68A7E3 has good binding affinity to C99 cells.

[0771] Example 5: Removal of CD16a antibody Y88A3 glycosylation sites

[0772] WO2022161314A1 details the discovery process of the CD16a antibody Y88A3. However, Y88A3 contains a glycosylation site NFT in CDR H1. Therefore, by site-directed mutagenesis (Anal Biochem.1989;179:309-311,DOI:10.1016 / 0003-2697(89)90134-6), the N at position 28 was mutated to T and the T at position 30 was mutated to A to obtain the target molecule Y88A3 (N28T; T30A). The molecular affinities before and after mutation are shown in Table 6 (the affinity determination method is described in Example 3, and the proteins used are human CD16a V176 (Acro, catalog number CD8-H52H4); human CD16a F176 (Acro, catalog number CDA-H5220), human CD16b (Acro, catalog number CDB-H5222); cynomolgus monkey C16a V176 (Acro, catalog number CD8-H52H4); cynomolgus monkey CD16a F176 (Acro, catalog number FC6-C52H9) and cynomolgus monkey CD16b (Acro, FC6-C52H9).

[0773] Table 6: Affinity of Candidate Molecules

[0774] Example 6. Affinity maturation of Anti-CD16a parent Y88A3 (N28T; T30A)

[0775] Affinity testing using BLI technology revealed that the desugared Y88A3 (N28T; T30A) molecules exhibited low affinity (see Table 6). Therefore, affinity maturation was initiated to enhance the molecule's function. Y88A3 (N28T; T30A) affinity maturation was performed using yeast display technology. The main procedures included library construction, library screening, yeast clone identification, protein expression, monoclonal antibody property analysis, and in vitro functional identification. Mutations were performed on the three CDRs of the single-domain antibody, both individually and simultaneously, resulting in the construction of four affinity-matured libraries. The diversity of each library reached 2.98E+08. The screening process included magnetic bead sorting and flow cytometry sorting.

[0776] Magnetic bead screening: Based on the principle that cell surface antigens can bind to specific monoclonal antibodies linked to magnetic beads, magnetic bead sorting is widely used in early antibody screening. In this case, the screening used was the Miltenyi MACS system. The main steps are as follows: Yeast cells larger than 10 times the library volume were taken from the library, and four libraries were screened using 100 nM Biotin-Human CD16a-V176 (Acro Biosystems, CDA-H82E9) and incubated at room temperature for 30 minutes. The cells were washed 1-2 times with buffer (1×PBS, containing 1% bovine serum albumin), resuspended with an appropriate volume of buffer, and streptavidin microbeads (Miltenyi, 130-048-101) were added and incubated at 4°C for 15 minutes. Centrifuge to remove unbound magnetic beads and resuspend. Add cell solution to Miltenyi LS column to complete adsorption and elution process. Collect eluted yeast cells and place them in culture flasks containing culture medium. Incubate overnight at 30°C, followed by shaking induction at 20°C for 24 hours.

[0777] Flow cytometry sorting: The cells from the magnetically enriched library were further sorted using a flow cytometer. Yeast cells larger than 10 times the library volume were taken from the magnetically bead-sorted library and washed 1-2 times with buffer. The cells were then incubated at room temperature for 30 minutes with Biotin-Human CD16a V176 (Acro Biosystems, CDA-H82E9) and Anti Flag (sigma, F1804). The cells were then washed twice with buffer and mixed with buffer containing streptavidin (SA-PE, eBioscience, catalog number: 12-4317-87) and goat anti-mouse conjugated Alex Flour-647 (Thermo Fisher, A21235). The mixture was incubated at 4°C in the dark for 15 minutes. The cells were then washed twice with pre-chilled buffer and resuspended in 2 mL of buffer. The cells were then transferred to a separation tube with a filter. Cells were sorted using the MoFlo_XDP ultra-high-speed flow cytometry system. The sorted yeast cells were then grown overnight at 30°C and induced with shaking at 20°C for 24 hours. Since the antibody molecules in this project need to bind to human CD16a-V176, human CD16a-F176, and cyno CD16a, but not to human CD16b-NA1 and human CD16b-NA2, subsequent screening will use human CD16a-F176 (Acro Biosystems, CDA-H82E8), human CD16b-NA1 (Acro Biosystems, CDB-H82E4), human CD16b-NA2 (Acro Biosystems, CDB-H82Ea), and Cyno CD16a (Acro Biosystems, FC6-C82E0) antigens. Through cross-screening with multiple antigens, approximately 1,000 clones were identified at the yeast level. Based on the intensity of fluorescence signals and sequence diversity, about 60 candidate molecules were constructed for expression and identification.

[0778] In protein property identification, affinity analysis was first performed on 60 candidate molecules. Most of the molecules bound to Human CD16a-NA1 after affinity maturation. Simultaneously, some molecules were artificially combined based on the amino acid patterns identified in the combinatorial library. For example, CDR H1 and H2 of molecule BC1148-C1 and CDR H3 of BC1147-D5 were combined to obtain molecules AM1510 and Am1510-m1-6.

[0779] The obtained VHH antibody was fused with the Fc region of IgG1 (containing the LALA mutation based on SEQ ID NO:58) to obtain the VHH-Fc fusion protein. The nucleic acid encoding the fusion protein was transfected into HEK293 cells for expression and purification to obtain the corresponding molecule. The protein was expressed in HEK293 cells (Shanghai Baiying Biotechnology, B843701-B843707). The sequence is shown in Table 7.

[0780] The obtained molecules were subjected to ADCC experiments, and the experimental method is as follows:

[0781] 1. Preheat CTS medium (Gibco, 0870112DK) to 37°C. Take huPBMC (human peripheral blood mononuclear cell) (Miaoshun Biotechnology, PB100C-W) and quickly thaw it in a water bath. Slowly add the cells to 8ml of CTS medium (containing 1% DNase).

[0782] 2. Centrifuge at 300g for 8 minutes, remove the supernatant, resuspend in 25ml CTS, transfer to T175 culture flask, and incubate overnight at 37℃.

[0783] 3. Take the overnight cultured suspension cells, centrifuge at 300g / 8min, remove the supernatant, and adjust the cell density with CTS; mix ADCC effector cells and target cells at a ratio of 50:1 evenly into a 96-well clear plate (NUNC, 167008).

[0784] 4. Add the antibody drug, diluted 5 times in CTS medium starting from 2 nM, to the corresponding cell well plate and incubate at 37°C with 5% CO2 for 6 h.

[0785] 5. Centrifuge at 300g for 4 min, and transfer 50 μL of the supernatant to a 96-well transparent flat plate (NUNC, 167008).

[0786] 6. Thaw the prepared Substrate Mix, add 50 μL of Substrate Mix to each well, and let stand in the dark for 30 min. Take 50 μL of the pre-prepared LDH reagent (Promega, G1780) and add it to the corresponding well plate, and let stand at room temperature in the dark for 15-30 min.

[0787] 7. Take 50 μL of LDH Stop Solution (Promega, G1780), read the value at 490 nm, and detect it using a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0788] ADCC function was validated in NCI-H292 cells. The results are shown in Figure 2. The killing effect of AMG1510-m4 was similar to that of the original clone AM1510.

[0789] Table 7: Candidate molecular sequences of CD16a nanobodies

[0790] Table 8: Affinity Testing After Engineering Modification

[0791] Example 7. Expression of CDH17 in cells

[0792] Cell flow cytometry showed that CDH17 was highly expressed in colorectal cancer cells (Figure 3).

[0793] Cell flow cytometry experimental method:

[0794] 1. Resuspend cells (GP2d cells, C99 cells, HT55 cells, SW948 cells, CCK-81 cells, H508 cells, C2BBE1 cells, SW403 cells and SW837 cells, Nanjing Kebai; SW1463 cells, Chinese Academy of Sciences Cell Bank) in FACS buffer, and seed 100,000-200,000 cells into 96-well plates (Corning, CLS3799-50EA). Add 20 μg / ml EGFR antibody (Cetuximab) and 20 μg / ml CDH17 antibody (expressed in HEK293 cells, sequence derived from WO2018115231A2) to the cell wells and incubate at 4 degrees Celsius for 1 hour.

[0795] 2. Wash twice with PBS, add the prepared APC-anti-human Fc antibody (Biolegend, 410712) to the corresponding cell well plate, and incubate at 4 degrees Celsius for 1 hour.

[0796] 3. Wash twice with PBS and detect with FACS (BD, Celesta).

[0797] Figure 3 shows the expression levels of CDH17 and EGFR in different cells. The expression level of CDH17 is relatively high in GP2d cells and C99 cells, while the expression level of CDH17 is relatively low in SW837 cells. The expression level of EGFR is relatively high in HT55 cells, while the expression level of EGFR is relatively low in SW837 cells.

[0798] Example 8. Pairing CDH17 and EGFR can enhance EGFR-mediated tumor suppression.

[0799] This embodiment constructs an EGFR / CDH17 bispecific antibody and verifies its efficacy.

[0800] Experimental antibody: Zalutumumab: EGFR IgG1 monoclonal antibody molecule. The light chain antibody sequence is SEQ ID NO: 1, and the heavy chain sequence is SEQ ID NO: 84. The antibody expression method is as follows.

[0801] Zalu / CDH17v1: The CDH17 / EGFR bispecific antibody molecule is assembled into a bispecific antibody in the form of an innobody (Innovent Biologics patent WO2022143912A1) (two parents, parent A's Fc contains S364R, D399K mutations and LALA mutations, parent B's Fc contains K370S, K409D, Y349T mutations and LALA mutations). The CDH17v1 sequence is derived from patent WO2018115231A2.

[0802] Zalu / GP120: The EGFR / IgG1 bispecific antibody molecule is also assembled into a bispecific antibody in the form of an innobody. The GP120 antibody clone is B12 (Nature. 2008; 455:109).

[0803] Zalu / CDH17 (P007, also known as B117-7): The CDH17 / EGFR bispecific antibody molecule is assembled into a bispecific antibody in the form of an innobody (Innovent Biologics patent WO2022143912A1) (two parents, parent A's Fc contains S364R, D399K mutations and LALA mutations, parent B's Fc contains K370S, K409D, Y349T mutations and LALA mutations). The CDH17 sequence is derived from Hz68A7E3 in Example 4.

[0804] Table 9: Antibody Structure

[0805] Methods of antibody expression:

[0806] Plasmid construction:

[0807] The heavy and light chain sequences were synthesized by Genewiz and loaded into a vector containing either the Fc region or the light chain constant region of pcDNA3.1.

[0808] Protein expression:

[0809] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF#A14351-01). Cell density was checked one day before transfection (viability should be greater than 95%), and the medium was adjusted to 3 × 10⁶ cells / day with fresh Expi293F medium. 6 Continue culturing at 100 cells / ml, and adjust the cell density to 3×100 on the day of transfection. 6 Cells / ml

[0810] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, with a light-to-heavy chain plasmid ratio of 1:1, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 min, then gently pour the mixture into the Expi293F cell suspension while shaking, place the cells in a shaker and culture under the following conditions: 8% CO2, 36.5℃, 120 rpm.

[0811] After culturing for 16–18 hours, 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), 5 g / L glucose solution, and 2.2 mM Valproic acid sodium salt (Merk, Cat#P4543-100G) were added to the cell suspension. The mixture was gently stirred and incubated for another 7 days at 36.5°C and 120 rpm with 8% CO2. The cells were then harvested. The cell suspension was mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0812] Affinity chromatography for purification of target protein:

[0813] Affinity capture was performed using a HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the column, followed by 10-20 column volumes of distilled water to wash the tubing and column. The column was then equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell stock was passed through the column, and the column was washed with 10 column volumes of 1×PBS to remove non-specific binding proteins. The column was then rinsed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2M Tris, filtered for sterilization, and prepared for further ion exchange chromatography. In vitro reduction-oxidation:

[0814] The protein collection buffer obtained from affinity chromatography was mixed at a 1:1 molar ratio, and an appropriate amount of GSH was added. The pH of the reaction was adjusted to 8.0 with 2M Tris. The mixture was incubated overnight at room temperature. The reaction mixture was then transferred to PBS and stored at 4°C for later use.

[0815] Ion exchange chromatography purification of bispecific antibodies:

[0816] A Mono S 5 / 50GL ion exchange chromatography column (from GE Healthcare) was used and placed in an AKTApure system (from GE Healthcare). The AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column was detoxified with 0.5M NaOH for 2 hours, followed by rinsing the system and column with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20mM NaPO4, pH 6.0) until conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was reequilibrated with 5 column volumes of loading buffer. Linear elution was performed using a gradient of 0-50% elution buffer (20mM NaPO4, 1M NaCl, pH 6.0) for a total of 30 column volumes. Samples were collected based on UV absorbance.

[0817] The purity of samples collected from each fraction was determined using size exclusion chromatography (SEC). Samples from fractions with a purity greater than 95% were pooled based on the SEC results. The purified bispecific antibody solution was centrifuged in 15 ml ultrafiltration centrifuge tubes at 4500 rpm for 30 minutes. The protein was diluted with PBS, and centrifugation was repeated at 4500 rpm for 30 minutes. This process was repeated several times, changing the buffer solution each time. The antibodies were pooled after each buffer change, and the antibody concentration was determined. Further qualitative and quantitative analysis of the bispecific antibody composition and content was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0818] In vitro tumor cell proliferation inhibition assay method:

[0819] Experimental cells: C2BBE1 cells, C99 cells, H508 cells, CCK-81 cells, GP2d cells, SW837 cells, and SW403 cells (Nanjing Kebai).

[0820] 1. 3000 cells / 100ul were seeded into 96-well white plates (NUNC, 136101) for 2D cell culture.

[0821] 2. The initial concentration of antibody molecules was 125 nM. It was diluted 5 times in the culture medium, added to the corresponding well plates, mixed well, and incubated at 37°C in a 5% CO2 incubator for four days.

[0822] 3. After continuous culture for 4 days in the proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells, and the cells were shaken at room temperature in the dark for 20 minutes before being directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0823] In vitro tumor cell proliferation inhibition experiments were conducted against the above-mentioned antibodies. The results (Figure 4) showed that the Zalu / CDH17 bispecific antibody had a superior EGFR-mediated tumor inhibition effect in CRC-KRAS wild-type and KRAS mutant cell lines compared with Zalutammumab monoclonal antibody.

[0824] Example 9. The structurally optimized CDH17 / EGFR / CD16A exhibits superior ADCC killing effect.

[0825] The EGFR / CDH17 bispecific antibody in Example 8 can be combined with the CD16A antibody in Example 6 to form a trispecific antibody, thereby enhancing the killing effect of Antibody-dependent cell cytotoxicity (ADCC).

[0826] The EGFR / CDH17 bispecific antibody molecule from Example 8 and the CD16A clone Am1510-m4 from Example 6 were assembled in innobody form (WO2022143912A1) to form six trispecific antibody molecules with different structures (Figure 5, structures P001-P006). The EGFR sequence was Zalutumumab, and the CDH17 sequence was derived from Hz68A7E3 from Example 4. The plasmid construction, protein expression, and purification methods were as described in Example 8, except that the low-fucose antibody was expressed in FUT8 knockout CHO cells.

[0827] The construction of molecules P001 to P008 (Figure 5) is as follows:

[0828] P001: EGFR / CDH17 bispecific antibody, low fucose. The antibody is expressed on FUT8 knockout CHO cells.

[0829] P002: AMG1510-m4 via a (G) 10 The adapter connects to the N-terminus of the full-length anti-CDH17 / EGFR antibody heavy chain.

[0830] P003: AMG1510-m4 via a (G) 10 The adapter connects to the N-terminus of the light chain of the full-length anti-CDH17 / EGFR antibody.

[0831] P004: AMG1510-m4 via a (G) 10The adapter connects to the C-terminus of the full-length anti-CDH17 / EGFR antibody heavy chain.

[0832] P005: AMG1510-m4 via a (G) 10 The adapter connects to the C-terminus of the full-length anti-CDH17 / EGFR antibody light chain.

[0833] P006: AMG1510-m4 via a (G) 10 The adapter connects the CDH17 and EGFR antibody Fab, and connects AMG1510-1 and antibody Fc through a (G)5 adapter.

[0834] P007: EGFR / CDH17 bispecific antibody, without CD16A. Fc has IgG1-LALA mutation. Sequence is identical to Zalu / CDH17 in Example 8 (Table 9).

[0835] P008: EGFR / CDH17 bispecific antibody, CD16A-free. Fc free of IgG1-LALA mutation. Sequence identical to P001. Antibody expressed on Expi293F cells.

[0836] The ADCC effects of the above eight molecules (Figure 5) were verified in SW837 cells, GP2d cells, and SW403 cells (Nanjing Kebai). The specific experimental methods are as follows:

[0837] 1. Seed the target cells overnight, 10,000 cells / well. Preheat CTS medium (Gibco, 0870112DK) to 37°C. Take huPBMCs (human peripheral blood mononuclear cells) (Miaoshun Biotechnology, PB100C-W) and quickly thaw them in a water bath. Slowly add the cells to 8 ml of CTS medium (containing 1% DNase).

[0838] 2. Centrifuge at 300g for 8 minutes, remove the supernatant, resuspend in 25ml CTS, transfer to T175 culture flask, and incubate overnight at 37℃.

[0839] 3. Take the overnight cultured suspension cells, centrifuge at 300g / 8min, remove the supernatant, and adjust the cell density with CTS; add ADCC effector cells (25μL / well) to a 96-well clear plate (NUNC,167008) with pre-coated target cells at an effector-to-target ratio of (30:1).

[0840] 4. The antibody molecule was initially diluted at a concentration of 25 nM, and then diluted 5-fold in culture medium. The diluted solutions were added to the corresponding cell culture plates and incubated at 37°C with 5% CO2 for 6 hours.

[0841] 5. Centrifuge at 300g for 4 min, and transfer 50 μL of the supernatant to a 96-well transparent flat plate (NUNC, 167008).

[0842] 6. Thaw the prepared Substrate Mix, add 50 μL of Substrate Mix to each well, and let stand in the dark for 30 min. Take 50 μL of the pre-prepared LDH reagent (Promega, G1780) and add it to the corresponding well plate, and let stand at room temperature in the dark for 15-30 min.

[0843] 7. Take 50 μL of LDH Stop Solution (Promega, G1780), read the value at 490 nm, and detect it using a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0844] The results showed that P006 exhibited superior activity compared to other structures in terms of EC50 and maximum toxicity (see Figure 6).

[0845] Table 10: The structure and sequence of the P001-6 molecule are as follows:

[0846] Example 10. In vitro tumor proliferation inhibition effect of P006

[0847] The following cells were used in an in vitro tumor cell proliferation inhibition experiment:

[0848] Wild-type KRAS colorectal cancer cells: C99, HT55, H508 (Nanjing Kebai)

[0849] KRAS-G12 mutant colorectal cancer cells: GP2D, SW1463, SW403 (Nanjing Kebai)

[0850] In vitro tumor cell proliferation inhibition assay method:

[0851] 1. 3000 cells / 100ul were seeded into 96-well white plates (NUNC, 136101) for 2D cell culture.

[0852] 2. The initial concentration of antibody molecules is 125 nM. Dilute the antibody molecules in the culture medium in a 5-fold gradient, add them to the corresponding cell well plates, mix well, and incubate at 37°C in a 5% CO2 incubator for 4 days.

[0853] 3. After continuous culture for 4 days in the proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells, and the cells were shaken at room temperature in the dark for 20 minutes before being directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0854] The results showed that P006 exhibited superior proliferation-inhibiting activity compared to cetuximab in both wild-type KRAS and KRAS-G12 mutant colorectal cancer cell models (Figures 7-8).

[0855] Example 11. ADCC in vitro pharmacodynamic activity of P006 antibody

[0856] ADCC experimental method:

[0857] The following cells were used in the experiment:

[0858] Wild-type KRAS colorectal cancer cells: C99, HT55, H508 (Nanjing Kebai)

[0859] KRAS-G12 mutant colorectal cancer cells: GP2D, SW837, SW403 (Nanjing Kebai)

[0860] 1. Seed the target cells overnight, 10,000 cells / well. Preheat CTS medium (Gibco, 0870112DK) to 37°C. Take huPBMCs (human peripheral blood mononuclear cells) (Miaoshun Biotechnology, PB100C-W) and quickly thaw them in a water bath. Slowly add the cells to 8 ml of CTS medium (containing 1% DNase).

[0861] 2. Centrifuge at 300g for 8 minutes, remove the supernatant, resuspend in 25ml CTS, transfer to T175 culture flask, and incubate overnight at 37℃.

[0862] 3. Take the overnight cultured suspension cells, centrifuge at 300g / 8min, remove the supernatant, and sort the NK cells using the NK Cell Isolation Kit (MILTENYI BIOTEC, 130-092-657). Adjust the cell density using CTS. Add 25μL / well of NK cells to a 96-well clear plate (NUNC, 167008) pre-coated with target cells at a ratio of 10:1 (NK cell (effect cell) to target cell).

[0863] 4. The antibody molecule was initially diluted at a concentration of 25 nM, and then diluted 5-fold in culture medium. The diluted solutions were added to the corresponding cell culture plates and incubated at 37°C with 5% CO2 for 6 hours.

[0864] 5. Centrifuge at 300g for 4 min, and transfer 50 μL of the supernatant to a 96-well transparent flat plate (NUNC, 167008).

[0865] 6. Thaw the prepared Substrate Mix, add 50 μL of Substrate Mix to each well, and let stand in the dark for 30 min. Take 50 μL of the pre-prepared LDH reagent (Promega, G1780) and add it to the corresponding well plate, and let stand at room temperature in the dark for 15-30 min.

[0866] 7. Take 50 μL of LDH Stop Solution (Promega, G1780), read the value at 490 nm, and detect it using a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0867] The results showed that P006 exhibited superior ADCC killing effects compared to cetuximab in both wild-type KRAS and KRAS-G12 mutant colorectal cancer cell models (Figures 9-10).

[0868] Example 12. Synergistic effect of P006 antibody combined with KRAS small molecule inhibitor

[0869] Experimental methods:

[0870] In vitro tumor cell proliferation inhibition assay method:

[0871] 1. 3000 cells / 100ul were seeded into 96-well white plates (NUNC, 136101) for 2D cell culture.

[0872] 2. Prepare Fulzerasib (Innovent Biologics) groups with a starting concentration of 125 nM and a 5-fold serial dilution in the culture medium, and Fulzerasib or MRTX1133 (MCE, HY-134813A) with a fixed final concentration of 125 nM in combination with P006. Add them to the corresponding well plates, mix well, and incubate at 37℃ in a 5% CO2 incubator for 4 days.

[0873] 3. After continuous culture for 4 days in the proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells, and the cells were shaken at room temperature in the dark for 20 minutes before being directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0874] The results showed that P006 of the present invention has a synergistic effect on killing tumors with the KRAS-G12C inhibitor Fulzerasib or the KRAS-G12D inhibitor MRTX1133 (Figure 11).

[0875] Example 13. In vitro safety study of P006 antibody drug

[0876] EGFR is highly expressed in normal skin cells, which leads to certain skin toxicity of EGFR monoclonal antibodies such as cetuximab. Clinically, patients may experience pustular rashes, paronychia, and dry and itchy skin. This affects the quality of life and treatment adherence of cancer patients and also limits the upper limit of drug use.

[0877] CDH17 is highly expressed in colorectal cancer with a near 100% positivity rate. However, in normal tissues, CDH17 is expressed only in the digestive tract, and its expression is limited to the tightly packed sidewalls of intestinal epithelial cells, rather than at the apical or apical ends accessible to antibodies. This reduces the damage to normal tissues caused by CDH17 antibodies. Therefore, the expression of EGFR and CDH17 in normal tissues is staggered. The combination of CDH17 / EGFR bispecific antibodies can reduce non-specific killing of normal tissues and enhance tumor cell recognition.

[0878] We selected human squamous cell carcinoma cell line (A431, ATCC) and human keratinocytes (HaCat, Cell lines service) as adverse reaction study models for P006 compared with cetuximab, and explored the safety of P006 in vitro.

[0879] First, the expression of CDH17 and EGFR in A431 cells and HaCaT cells was detected using the following methods:

[0880] 1. Resuspend the cells in FACS buffer, and seed 100,000-200,000 cells into a 96-well plate (Corning, CLS3799-50EA). Add 20 μg / ml EGFR antibody (Cetuximab) and 20 μg / ml CDH17 antibody (expressed on HEK293 cells, sequence derived from WO2018115231A2) to the cell wells and incubate at 4°C for 1 hour.

[0881] 2. Wash twice with PBS, add the prepared APC-anti-human Fc antibody (Biolegend, 410712) to the corresponding cell well plate, and incubate at 4 degrees Celsius for 1 hour.

[0882] 3. Wash twice with PBS, and detect the expression levels of (BD, Celesta) EGFR and CDH17 using FACS.

[0883] The results are shown in Figure 12, which shows that both A431 and HaCat skin cells overexpress EGFR but do not express CDH17.

[0884] Subsequently, the inhibitory effects of P006 and Cetuximab on the proliferation of tumor cells in A431 and HaCaT cells were examined, using the following methods:

[0885] 1. 3000 cells / 100ul were seeded into 96-well white plates (NUNC, 136101) for 2D cell culture.

[0886] 2. The initial concentration of antibody molecules is 125 nM. Dilute the antibody molecules in the culture medium in a 5-fold gradient, add them to the corresponding well plates, mix well, and incubate at 37°C in a 5% CO2 incubator for 4 days.

[0887] 3. After continuous culture for 4 days in the proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells, and the cells were shaken at room temperature in the dark for 20 minutes before being directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0888] The test results are shown in Figure 13, which shows that P006 has a significantly lower killing effect on skin cells than cetuximab.

[0889] Example 14. In vivo pharmacodynamic activity of P006 antibody

[0890] To evaluate the in vivo efficacy of the P006 antibody, wild-type KRAS NCI-H508 colorectal cancer tumor cells (Nanjing Kebai) were subcutaneously inoculated into NOG immunodeficient mice (Vitaliva). The tumors were allowed to grow to approximately 200 mm. 3 They were then divided into groups and given the drugs to be tested.

[0891] The specific method is as follows:

[0892] 1. NCI-H508 tumor cells (Nanjing Kebai) were cultured using standard cell passage methods. After reaching a sufficient cell number, the cells were digested, centrifuged, and collected. The cells were resuspended in PBS and then mixed with Matrigel Matix (Corning, 356231) in an equal proportion to prepare a cell concentration of 20 × 10⁻⁶ cells / mL. 6 Cell suspension of cells / ml;

[0893] 2. Take 0.2 ml of cell suspension and subcutaneously inoculate it into the right abdominal region of 6 to 8-week-old SPF-grade female NOG mice (Vitalliwa) to establish a tumor-bearing mouse model and monitor the growth of tumors after subsequent subcutaneous inoculation;

[0894] 3. When the subcutaneous tumor reaches a volume of approximately 200 mm 3Subsequently, the tumor-bearing mice were divided into groups of 6 mice each, so that the average tumor size of each group was similar. The mice were intraperitoneally injected with the test drug P006, the anti-human epidermal growth factor (EGFR) antibody Cetuximab, and the negative control IgG (GP120IgG1 monoclonal antibody, heavy chain sequence is SEQ ID NO:85, light chain sequence is SEQ ID NO:71, expression method is as shown in Example 8), at a dose of 1 mg / kg.

[0895] 4. Administer the medication twice a week for four weeks, monitoring tumor volume and body weight in mice twice a week and recording the data;

[0896] 5. Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = L * W² / 2. Body weight was measured using an electronic balance. Mice with tumor volumes exceeding 2000 mm³ or body weight loss exceeding 20% ​​were euthanized.

[0897] Tumor growth inhibition (TGI) = 100% * (mean tumor volume of IgG group – mean tumor volume of drug-treated group) / (mean tumor volume of IgG group – mean tumor volume of IgG group at the time of drug administration).

[0898] The results are shown in Figure 14. P006 demonstrated better antitumor efficacy than cetuximab. Cetuximab had a TGI of 57%, while P006 had a tumor inhibition rate of 107%.

[0899] Example 15. In vivo pharmacodynamic activity of P006 molecule combined with VEGF small molecule inhibitor

[0900] Small molecule inhibitors of VEGF can promote changes in the immune microenvironment, increase tumor infiltration of immune cells, enhance NK cell-mediated ADCC killing, and strengthen the tumor-killing effect of P006.

[0901] Experimental method for evaluating drug efficacy in xenograft tumors in mice:

[0902] B-NDG-hIL15 mice (Biocytogen) were subcutaneously inoculated with GP2D cell suspension, 4 × 10⁴ cells per cell line. 6 A tumor-bearing model was established using 1 mouse per mouse.

[0903] On the 3rd day after subcutaneous inoculation, PBMC cell suspension (Miaoshun Biotechnology) was injected at a dose of 4×10⁶ cells / day. 6 One mouse per subcutaneous inoculation; on day 9 post-inoculation, the average tumor volume in mice reached 116.47 mm. 3 -167.39mm 3At that time, 28 mice with tumors were selected and divided into 4 groups of 7 mice each: IgG, 10 mg / kg; P006, 10 mg / kg; Fruquintinib, 1 mg / kg; and P006, 10 mg / kg + Fruquintinib, 1 mg / kg combination therapy group.

[0904] Fruquinnib is administered by gavage, starting on day 9, once daily for 21 days without intervals. IgG and P006 are administered by intraperitoneal injection, twice a week for a total of 6 doses, on days 9, 13, 16, 20, 23, and 27.

[0905] Mouse body weight, maximum long axis (L), and maximum wide axis (W) of tumor tissue were monitored twice a week for 37 days. After the experiment, the relative tumor inhibition rate of each group of mice was calculated.

[0906] Tumor volume determination: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = L * W² / 2. Body weight was measured using an electronic balance. Mice with tumor volumes exceeding 2000 mm³ or body weight loss exceeding 20% ​​were euthanized. The TGI calculation method was the same as in Example 14.

[0907] Results from the GP2d in vivo model showed that the combination of P006 antibody and the VEGF small molecule inhibitor fruquintinib (MCE, HY-19912) had a synergistic tumor-killing effect (Figure 15). The TGI of fruquintinib was 36.6%, that of P006 was 41.3%, and that of the combination of P006 and fruquintinib was 63.0%. The tumor-suppressive effect of the combination of P006 and fruquintinib was stronger than that of fruquintinib or P006 monotherapy.

[0908] Example 16. P006 bsAb antibody pretoxicology in scorpion-eating monkeys

[0909] Preliminary species affinity data for P006 showed that the three targets exhibited affinity for cynomolgus monkeys comparable to that for humans, but did not bind to rodents. Therefore, the preclinical animal species associated with P006 was the cynomolgus monkey. Pre-toxicology studies of P006 were conducted exclusively on cynomolgus monkeys, with two dosage groups (50 and 150 mg / kg) administered intravenously, using two cynomolgus monkeys (Yunnan Jinjiekang Biotechnology) per group. Dosage was administered once a week for four weeks. The main endpoints included clinical observation, clinicopathology, cytokines, immunophenotype, and gross and histopathological examinations.

[0910] In the pre-toxicology study of P006 monkeys, no animals experienced intolerance-related deaths. At a dose of 50 mg / kg, two animals showed mild loose stools or soft stools during administration, and one animal showed a slight decrease in body weight. Histopathology revealed mild inflammatory cell infiltration in the kidneys in two animals and mild chronic inflammation of the gastric mucosa in one animal. No other significant abnormalities were observed. In the 150 mg / kg dose group, two animals experienced varying degrees of diarrhea, decreased body weight (20%), and decreased food intake during administration. One animal showed elevated globulin and decreased albumin / globulin ratio after the third and fourth administrations, which were considered to be related to diarrhea and decreased food intake. Elevated C-reactive protein and fibrinogen levels suggested inflammation. All of these indicators returned to normal before autopsy. Gross autopsy revealed a small thymus in one animal. Histopathology mainly showed mild to mild mucosal inflammation / necrosis in the stomach, rectum, and duodenum, and mild to mild renal tubular dilation in the kidneys. One animal showed severe cortical lymphopenia in the thymus, which could not be ruled out as being related to individual differences. In summary, no serious toxicity was found in the pretoxicology studies of P006, and the maximum non-serious toxic dose (HNSTD) was set at 150 mg / kg.

[0911] The study also investigated marketed EGFR-targeting cetuximab. Its application materials disclosed that in a 39-week Good Clinical Practice (GLP) monkey toxicology study, half of the animals died of sepsis at a high dose of 75 mg / kg, primarily due to abnormalities in ocular and skin toxicity, liver function, and albumin levels caused by diarrhea. Overall, P006 showed better tolerable doses than cetuximab.

[0912] Sequence information:

Claims

1. An anti-CDH17 antibody or antigen-binding fragment thereof, comprising three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 21, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 16; or three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 25, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO:

26.

2. An anti-CDH17 antibody or antigen-binding fragment thereof, comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or 27, SEQ ID NO: 24, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, respectively.

3. The anti-CDH17 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region a) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21; or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21; or b) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25; or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25; and / or the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region a) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16; or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16; or b) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26; or comprises or consists of the amino acid sequence set forth in SEQ ID NO:

26. a) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; or b) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 26; or comprises or consists of the amino acid sequence set forth in SEQ ID NO:

26.

4. An anti-CDH17 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region, VH, and a light chain variable region, VL, wherein a) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 16; or b) the heavy chain variable region comprises or consists of a VH set forth in SEQ ID NO: 25 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 26; or c) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16; or d) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO:

26.

5. The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a heavy chain constant region that is IgGl, IgG2, IgG3, or IgG4, e.g., a constant region of human IgGl, IgG2, IgG3, or IgG4, e.g., the heavy chain constant region a) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 55; and / or b) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 55; and / or the light chain constant region is a lambda or Kappa light chain constant region, e.g., a Kappa light chain constant region, e.g., a human lambda or Kappa light chain constant region, e.g., the light chain constant region a) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43 or 56; or b) comprises or consists of the amino acid sequence of SEQ ID NO: 43 or 56.

6. The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody is a monoclonal antibody; and / or the antibody is a humanized antibody or a chimeric antibody.

7. The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antigen-binding fragment is an antibody fragment selected from a Fab, a Fab’, a Fab’-SH, an Fv, a single-chain antibody (e.g., scFv), a (Fab’)2, a dAb (domain antibody), a diabody, or a linear antibody.

8. The anti-CDH17 antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the anti-CDH17 antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity for CDH17 and a binding specificity for one or more other antigens; optionally, the other antigen is EGFR; or the other antigen is EGFR and CD16A.

9. A multispecific antibody comprising a first antigen-binding region that specifically binds to EGFR and a second antigen-binding region that specifically binds to CDH17, and optionally a third antigen-binding region that specifically binds to CD16A; optionally, the multispecific antibody is a bispecific antibody and it comprises a first antigen-binding region that specifically binds to EGFR and a second antigen-binding region that specifically binds to CDH17; or the multispecific antibody is a trispecific antibody and it comprises a first antigen-binding region that specifically binds to EGFR and a second antigen-binding region that specifically binds to CDH17, and a third antigen-binding region that specifically binds to CD16A.

10. The multispecific antibody of claim 9, wherein the second antigen-binding region specifically binds to CDH17 and comprises a VH and a VL, wherein a) the VH comprises HCDR1, HCDR2, HCDR3 as defined in claim 1 or 2, and the VL comprises LCDR1, LCDR2, and LCDR3 as defined in claim 1 or 2; or b) The VH comprises three complement-determining regions (HCDRs) of heavy chain variable regions, HCDR1, HCDR2 and HCDR3, and the VL comprises three complement-determining regions (LCDRs) of light chain variable regions, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:97; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:98; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:99; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:100; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:101; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:

102. c) The VH and VL are the VH and VL as defined in claim 3 or 5; or d) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:88, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:

89.

11. The multispecific antibody of claim 9 or 10, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein The HCDR1, HCDR2, and HCDR3 are the heavy chain variable regions HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, and the LCDR1, LCDR2, and LCDR3 are the light chain variable regions LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:2; or the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:3, the LCD R2 contains or is composed of the amino acid sequence of SEQ ID NO:4, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:5; or The first antigen-binding region comprises VH and VL, wherein VH comprises or is composed of the sequence shown in SEQ ID NO:7; and VL comprises or is composed of the sequence shown in SEQ ID NO:

2.

12. The multispecific antibody according to any one of claims 9-11, wherein the second antigen-binding region is a Fab that specifically binds to CDH17; and / or the first antigen-binding region is a Fab that specifically binds to EGFR; Optionally, the Fab serving as the first antigen-binding region or the second antigen-binding region comprises CH1, wherein the CH1 is CH1 derived from IgG1, IgG2, IgG3, or IgG4, preferably CH1 derived from IgG1, for example, CH1 (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 44; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 44; and / or said Fab as first antigen binding region or second antigen binding region comprises a light chain constant region, wherein said light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, for example said Kappa light chain constant region (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 43; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 43; or said Lambda light chain constant region (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 56; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

56.

13. The multispecific antibody of claim 12, wherein the Fab as first antigen binding region comprises a Fab heavy chain and a Fab light chain, wherein said Fab heavy chain comprises (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 49; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 49; and said Fab light chain comprises (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1 ; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

1.

14. The multispecific antibody of claim 12 or 13, wherein the second antigen binding region is a Fab of an antibody as defined in any one of claims 1 to 10; optionally, the Fab as second antigen binding region comprises a Fab heavy chain and a Fab light chain, wherein said Fab heavy chain comprises (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 50; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 50; and the Fab light chain comprises (i) an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or consists of, the amino acid sequence of SEQ ID NO: 15; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 15; or the Fab heavy chain comprises (i) an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or consists of, the amino acid sequence of SEQ ID NO: 90; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 90; and the Fab light chain comprises (i) an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or consists of, the amino acid sequence of SEQ ID NO: 92; (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

92.

15. The multispecific antibody of any one of claims 9-13, wherein the multispecific antibody is an IgG-like multispecific antibody comprising an Fc dimer, wherein the two Fc regions making up the Fc dimer are identical or different, optionally the Fc regions are human IgGl, IgG2, IgG3, or IgG4 Fc, optionally the Fc regions comprise (i) an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or consists of, the amino acid sequence selected from the group consisting of SEQ ID NO: 57 or 58; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 57 or 58; optionally the two Fc regions are different, wherein mutations are introduced in the first Fc region and the second Fc region based on the Innobody technology, promoting heterodimerization of the first Fc region and the second Fc region; preferably wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S, and K409D mutations; optionally the first and / or second Fc region further comprises L234A / L235A mutations; optionally the two Fc regions are different, and wherein a) one Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47, and the other Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48; b) one Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and the other Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87; c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 86 and comprises the mutations S364R and D399K and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 87 and comprises the mutations Y349T, K370S and K409D; or d) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 47 and comprises the mutations S364R and D399K and the L234A / L235A mutations and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 48 and comprises the mutations Y349T, K370S and K409D and the L234A / L235A mutations.

16. The multispecific antibody of any one of claims 9-14, wherein the third antigen binding region is a VHH that specifically binds CD16A; optionally, the VHH antibody that specifically binds CD16A comprises three complementarity determining regions (CDRs) contained in a VHH set forth in any one of SEQ ID NOs: 11, 28, 32, 34, 37, 38, 39, 40, or 41; or the VHH that specifically binds CD16A comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:12, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14 or 36 or 42; (ii) VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or 33, VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, and VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31; or (iii) VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36 or 14; or the VHH that specifically binds CD16A comprises or consists of a heavy chain variable region, the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40, or 41; or (ii) comprises or consists of the amino acid sequence set forth in any one of SEQ ID NO: 11, 28, 32, 34, 37, 38, 39, 40, or 41.

17. The multispecific antibody of any one of claims 9-16, which is a bispecific antibody, and comprises or consists of: a heavy chain A comprising or consisting of, from N- to C-terminal: a first Fab heavy chain variable region - a heavy chain constant region CH1 - a first Fc region; a light chain a comprising or consisting of, from N- to C-terminal: a first Fab light chain variable region - a light chain constant region; a heavy chain B comprising or consisting of, from N- to C-terminal: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region; a light chain b comprising or consisting of, from N- to C-terminal: a second Fab light chain variable region - a light chain constant region; wherein the first antigen binding region is a first Fab that specifically binds EGFR, and the second antigen binding region is a second Fab that specifically binds CDH17; the heavy chain variable region and the light chain variable region of the first Fab are as defined in claim 11; and the heavy chain variable region and the light chain variable region of the second Fab are as defined in claim 10; or the heavy chain and the light chain of the first Fab are as defined in claim 13; the heavy chain and the light chain of the second Fab are as defined in claim 14; the heavy chain constant region CH1 and the light chain constant region are as defined in claim 12; and the first Fc region and the second Fc region are as defined in claim 15.

18. The multispecific antibody of claim 17, wherein the heavy chain A comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto; the light chain a comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto; the heavy chain B comprises or consists of the amino acid sequence set forth in SEQ ID NO: 68, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto; and / or the light chain b comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto; or the heavy chain A comprises an amino acid sequence as set forth in SEQ ID NO: 70, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; the light chain a comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; the heavy chain B comprises an amino acid sequence as set forth in SEQ ID NO: 91, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and / or the light chain b comprises an amino acid sequence as set forth in SEQ ID NO: 92, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; or the heavy chain A comprises an amino acid sequence as set forth in SEQ ID NO: 74, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; the light chain a comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; the heavy chain B comprises an amino acid sequence as set forth in SEQ ID NO: 80, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and / or the light chain b comprises an amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence.

19. The multispecific antibody of any one of claims 9-18, which is a trispecific antibody comprising a first antigen binding region first Fab that specifically binds EGFR, a second antigen binding region second Fab that specifically binds CDH17, and a third antigen binding region first and second VHH that specifically binds CD16A, and optionally comprising a first and a second Fc region; optionally, wherein the first and second VHH are linked to the N-terminus of the Fab heavy chain of the first and second Fab, respectively, or are inserted between the C-terminus of the Fab heavy chain of the first and second Fab and the Fc region, respectively, or are linked to the N- or C-terminus of the Fab light chain of the first and second Fab, respectively, or are linked to the C-terminus of the first and second Fc region, respectively, optionally, wherein the first and second VHH are identical or different; ​ wherein the heavy chain variable region and the light chain variable region of the first Fab are as defined in claim 11; and the heavy chain variable region and the light chain variable region of the second Fab are as defined in claim 10; or the heavy chain and the light chain of the first Fab are as defined in claim 13; the heavy chain and the light chain of the second Fab are as defined in claim 14; the first VHH and the second VHH are as defined in claim 16, respectively; and the first Fc region and the second Fc region are as defined in claim 15.

20. The multispecific antibody of claim 19, comprising or consisting of: a heavy chain A comprising or consisting of, from N-terminus to C-terminus: a first Fab heavy chain variable region - a heavy chain constant region CH1 - a first Fc region; a light chain a comprising or consisting of, from N-terminus to C-terminus: a first Fab light chain variable region - a light chain constant region; a heavy chain B comprising or consisting of, from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region; a light chain b comprising or consisting of, from N-terminus to C-terminus: a second Fab light chain variable region - a light chain constant region; wherein the first Fab specifically binds to a first antigen, and the second Fab specifically binds to a second antigen; wherein the heavy chain A and the heavy chain B further comprise a first VHH and a second VHH, respectively, wherein the C-terminus of the first VHH and the second VHH is connected to the N-terminus of the first Fab heavy chain variable region and the second Fab heavy chain variable region, respectively, via or without a linker; or the N-terminus of the first VHH and the second VHH is connected to the C-terminus of the first Fc region and the second Fc region, respectively, via or without a linker; or wherein the light chain a and the light chain b further comprise a first VHH and a second VHH, respectively, wherein the C-terminus of the first VHH and the second VHH is connected to the N-terminus of the first Fab light chain variable region and the second Fab light chain variable region, respectively, via or without a linker, or wherein the N-terminus of the first VHH and the second VHH is connected to the C-terminus of the first Fab light chain variable region and the second Fab light chain variable region, respectively, via or without a linker; wherein the heavy chain variable region and the light chain variable region of the first Fab are as defined in claim 11; and the heavy chain variable region and the light chain variable region of the second Fab are as defined in claim 10; or the heavy chain and the light chain of the first Fab are as defined in claim 13; the heavy chain and the light chain of the second Fab are as defined in claim 14; the first VHH and the second VHH are as defined in claim 16, respectively; the heavy chain constant region CH1 and the light chain constant region are as defined in claim 12; and the first Fc region and the second Fc region are as defined in claim 15.

21. The multispecific antibody of claim 20, wherein the first VHH and the second VHH are each connected via a linker, the linker comprising (G) n wherein n = any integer between 1 and 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably 5 or 10, such as the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45 or 46.

22. The multispecific antibody of claim 20 or 21, wherein the heavy chain A comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and the heavy chain B comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and the light chain a comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and the light chain b comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 78, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. The heavy chain A comprises the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; The heavy chain B comprises an amino acid sequence as set forth in SEQ ID NO: 81, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and The heavy chain B comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; or The heavy chain A comprises the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; The light chain a comprises the amino acid sequence shown in SEQ ID NO: 78, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; The heavy chain B comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and The light chain b comprises the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; or The heavy chain A comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; The light chain a comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; The heavy chain B comprises the amino acid sequence shown in SEQ ID NO: 83, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and The light chain b comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; or The heavy chain A comprises the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; The light chain a comprises the amino acid sequence shown in SEQ ID NO: 79, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; the heavy chain B comprises or consists of the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and the light chain b comprises or consists of the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

23. The multispecific antibody of claim 19, comprising or consisting of: the heavy chain A comprises or consists of, from N- to C-terminal: a first Fab heavy chain variable region - a heavy chain constant region CH1 - a first VHH - a first Fc region, wherein the heavy chain constant region CH1 at its C-terminus is connected to the N-terminus of the first VHH and the first VHH at its C-terminus is connected to the N-terminus of the first Fc region, via or without a linker; the light chain a comprises or consists of, from N- to C-terminal: a first Fab light chain variable region - a light chain constant region; the heavy chain B comprises or consists of, from N- to C-terminal: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second VHH - a second Fc region, wherein the heavy chain constant region CH1 at its C-terminus is connected to the N-terminus of the second VHH and the second VHH at its C-terminus is connected to the N-terminus of the second Fc region, via or without a linker; the light chain b comprises or consists of, from N- to C-terminal: a second Fab light chain variable region - a light chain constant region; wherein the first Fab heavy chain variable region and light chain variable region are as defined in claim 11 ; and the second Fab heavy chain variable region and light chain variable region are as defined in claim 10; or the first Fab heavy chain and light chain are as defined in claim 13; the second Fab heavy chain and light chain are as defined in claim 14; the first VHH and second VHH are each as defined in claim 16; the heavy chain constant region CH1 and the light chain constant region are as defined in claim 12; and the first Fc region and second Fc region are as defined in claim 15.

24. The multispecific antibody of claim 23, wherein the first or second VHH at its N-terminus is connected to the C-terminus of the first or second Fab heavy chain via a linker 1 and the first or second VHH at its C-terminus is connected to the N-terminus of the first or second Fc region via a linker 2; Optionally, the linker 1 or 2 comprises (G) n wherein n = any integer between 1 and 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5 or 10, for example linker 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45, and / or linker 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

46.

25. The multispecific antibody of claim 23 or 24, comprising or consisting of the heavy chain A, the heavy chain B, the light chain a and the light chain b, wherein the heavy chain A comprises or consists of the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; the light chain a comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; the light chain a comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; The heavy chain B comprises an amino acid sequence as set forth in SEQ ID NO: 20, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and / or The light chain b comprises an amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence.

26. A VHH antibody specifically binding to CD16A, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3 are the three complementarity determining regions (CDRs) contained in a VHH as set forth in SEQ ID NO: 11, 32, 34, 37, 38, 39, 40 or 41; or (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:12, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, VHH CDR3 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 14 or 36 or 42; (ii) VHH CDR1 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 33, VHH CDR2 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 30, and VHH CDR3 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 31; or (iii) VHH CDR1 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 35, VHH CDR2 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 13, and VHH CDR3 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 36 or 14.

27. The VHH antibody specifically binding to CD16A of claim 26, comprising or consisting of a heavy chain variable region, which (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 32, 34, 37, 38, 39, 40 or 41; or (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 32, 34, 37, 38, 39, 40 or 41.

28. A heavy chain antibody specifically binding to CD16A, comprising the VHH antibody specifically binding to CD16A of claim 26 or 27; optionally, the heavy chain antibody comprises a heavy chain comprising or consisting of the VHH antibody specifically binding to CD16A of claim 26 or 27 linked to an antibody heavy chain constant region or Fc region; optionally, the antibody heavy chain constant region is as defined in claim 5, or the Fc region is as defined in claim 15, optionally the Fc region further comprises the L234A / L235A mutation; Optionally, the heavy chain antibody comprises or consists of a heavy chain (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 60-67; or (ii) comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 60-67; Optionally, the heavy chain antibody comprises two heavy chains that are dimerized, wherein the two heavy chains are identical or different.

29. The VHH antibody specifically binding to CD16A of claim 26 or 27, or the heavy chain antibody specifically binding to CD16A of claim 28, wherein the antibody is a chimeric antibody or a humanized antibody.

30. A nucleic acid molecule comprising or consisting of a polynucleotide encoding any one of the chains of said antibody or antigen-binding fragment thereof of any one of claims 1-8 or multispecific antibody of any one of claims 9-25 or VHH antibody specifically binding to CD16A of any one of claims 26, 27 and 29, or heavy chain antibody specifically binding to CD16A of claim 28 or 29.

31. An expression vector comprising the nucleic acid molecule of claim 30, preferably the expression vector is pCDNA, such as pCDNA3.

1.

32. A host cell comprising the nucleic acid molecule of claim 30 or the expression vector of claim 31, preferably the host cell is prokaryotic or eukaryotic, such as a 293 cell or a CHO cell, such as an Expi293F cell.

33. A method of producing said antibody or antigen-binding fragment thereof of any one of claims 1-8 or multispecific antibody of any one of claims 9-25 or VHH antibody specifically binding to CD16A of any one of claims 26, 27 and 29, or heavy chain antibody specifically binding to CD16A of claim 28 or 29, the method comprising culturing a host cell of the nucleic acid molecule of claim 30 or the expression vector of claim 31 under conditions suitable for expression of the polypeptide chain of said antibody or antigen-binding fragment thereof, and optionally recovering said antibody from said host cell (or host cell culture medium).

34. An immunoconjugate comprising said antibody or antigen-binding fragment thereof of any one of claims 1-8 or multispecific antibody of any one of claims 9-25, such as an antibody-drug conjugate.

35. A pharmaceutical composition comprising said antibody or antigen-binding fragment thereof of any one of claims 1-8 or multispecific antibody of any one of claims 9-25 or VHH antibody specifically binding to CD16A of any one of claims 26, 27 and 29, or heavy chain antibody specifically binding to CD16A of claim 28 or 29, or immunoconjugate of claim 34, and optionally a pharmaceutical excipient.

36. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the multispecific antibody of any one of claims 9-25 or the VHH antibody specifically binding to CD16A of any one of claims 26, 27 and 29, or the heavy chain antibody specifically binding to CD16A of claim 28 or 29, or the immunoconjugate of claim 34, and one or more other therapeutic agents, e.g., wherein the therapeutic agent is a variety of therapeutic agents for treating a tumor, e.g., a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist), preferably, the small molecule drug is selected from a KRAS small molecule inhibitor, e.g., a KRAS-G12C inhibitor, such as sotorasib; or a KRAS-G12D inhibitor such as MRTX1133; or a VEGF small molecule inhibitor, e.g., a fynarid.

37. A method of preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the multispecific antibody of any one of claims 9-25 or the VHH antibody specifically binding to CD16A of any one of claims 26, 27 and 29, or the heavy chain antibody specifically binding to CD16A of claim 28 or 29, or the immunoconjugate of claim 34, or the pharmaceutical composition or formulation of claim 35, or the pharmaceutical combination of claim 36; optionally, wherein the tumor is a solid tumor or a hematological tumor, e.g., a CDH17 positive tumor or cancer, or a CDH17 and EGFR positive tumor or cancer, e.g., the tumor is a colorectal cancer, a colon cancer (e.g., colon cancer adenocarcinoma), or a rectal cancer, e.g., the tumor is a KRAS mutant tumor or cancer, e.g., a tumor or cancer having a KRAS G12 mutation (e.g., a G12D, G12C, or G12V mutation); optionally, the method further comprises administering in combination with another therapy, e.g., a treatment modality (e.g., surgery or radiotherapy) and / or another therapeutic agent, preferably, the therapeutic agent is a variety of therapeutic agents for treating a tumor, e.g., a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist), preferably, the small molecule drug is selected from a KRAS small molecule inhibitor, e.g., a KRAS-G12C inhibitor, such as sotorasib; or a KRAS-G12D inhibitor such as MRTX1133; or a VEGF small molecule inhibitor, e.g., a fynarid.

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