Antigen-binding constructs targeting her2 and uses

Abstract

To provide bispecific antigen-binding constructs that bind HER2 with reduced aggregation of the antigen-binding constructs, and uses.SOLUTION: Provided are antigen-binding constructs that target HER2 with specific sequences, nucleic acids encoding them, vectors comprising the nucleic acids, cells comprising the vectors, pharmaceutical compositions comprising them, and uses thereof, such as in treating a subject having a HER2 expressing tumor, killing HER2 expressing tumor cells, or inhibiting the growth of HER2 expressing tumor cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antigen-binding constructs, and more particularly to antigen-binding constructs that target HER2 and uses thereof. [Background technology]

[0002] Her2 (human epidermal growth factor receptor 2), also known as ErbB-2 (receptor tyrosine-protein kinase erbB-2), is a member of the human epidermal growth factor receptor (HER / EGFR / ERB) family, which includes EGFR (ErbB-1), Her2 / c-neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). The Her2 protein contains extracellular ligand-binding domains (domains I-IV), a transmembrane domain, and an intracellular domain. The ligand for Her2 is unknown. Her2 forms heterodimers with one of three other receptors (ErbB-1, ErbB-3, and ErbB-4), and dimerization induces autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, resulting in the activation of MAPK (mitogen-activated protein kinase), PI3K / Akt (phosphoinositide 3-kinase), and PKC (protein It activates various signaling pathways, including kinase C (protein kinase C) and STAT (signal transducer and activator of transcription). Amplification or overexpression of the Her2 gene plays an important role in the development and progression of some invasive breast cancers, and 15-30% of breast cancer patients are Her2-positive, making Her2 an important biomarker and therapeutic target in breast cancer patients. Her2 is also overexpressed in 7-34% of gastric cancer patients and in 30% of salivary gland duct carcinomas.

[0003] Several Her2-targeting antibody drugs are already on the market for treating Her2-positive tumors. These include Roche's monoclonal antibody drug trastuzumab (Herceptin®), which targets Her2 extracellular domain IV (ECD4), Roche's pertuzumab (Perjeta®), which targets Her2 extracellular domain II (ECD2), and Roche's ADC drug, T-DM1® (ado-trastuzumab emtansine). The mechanisms of action of these monoclonal antibody drugs include: 1) downregulation of Her2 signaling, 2) formation of a signaling heterodimer epitope by binding to Her2, 3) internalization of Her2, and 4) antibody-dependent cellular cytotoxicity (ADCC) of the monoclonal antibody. Monospecific targeting does not target other target epitopes that may be involved in signal transduction and pathogenesis, which can lead to drug resistance and immune evasion.

[0004] Antibody aggregation poses challenges in expression and / or purification, immunogenicity, toxicity, degradation, reduced affinity, or loss of activity after storage. Therefore, reducing antibody aggregation is necessary for industrial applications. Since scFvs are single-chain molecules, they are prone to aggregation, and when aggregated, dimers (diabodies), trimers (triabodies), and tetrabodies are formed. Summary of the Invention

[0005] In accordance with the present invention, an antigen-binding construct is provided, which exhibits reduced aggregation of the antigen-binding construct. The invention further provides uses of the antigen-binding constructs.

[0006] In one aspect of the present invention, there is provided an antigen-binding construct comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, wherein the first antigen-binding fragment is an scFv, wherein the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 of the Kabat numbering system, preferably an E amino acid after the mutation, and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 of the Kabat numbering system, preferably a Y amino acid after the mutation.

[0007] Alternatively, the present invention provides an antigen-binding construct comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, wherein the first antigen-binding fragment is an scFv, wherein the heavy chain variable region of the first antigen-binding fragment contains an amino acid at position 30 of the Kabat numbering system that is K or E, and / or the light chain variable region of the first antigen-binding fragment contains an amino acid at position 53 of the Kabat numbering system that is F or Y, provided that when the amino acid at position 30 of the heavy chain variable region of the first antigen-binding fragment is K of the Kabat numbering system, the amino acid at position 53 of the Kabat numbering system of the light chain variable region of the first antigen-binding fragment is not F.

[0008] In some embodiments, the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system, and / or the light chain variable region of the first antigen-binding fragment comprises a F53Y mutation according to the Kabat numbering system. In some specific embodiments, the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system. In some specific embodiments, the light chain variable region of the first antigen-binding fragment comprises a F53Y mutation according to the Kabat numbering system. In some embodiments, the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system, and the light chain variable region of the first antigen-binding fragment comprises a F53Y mutation according to the Kabat numbering system.

[0009] In some embodiments, the first antigen-binding fragment comprises a group of CDRs selected from: i. It comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. ii. It comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. iii. It comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. iv. It comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. v. Comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. vi. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 are each independently Each of them comprises an amino acid sequence having at least 80% identity with the amino acid sequences of SEQ ID NOs: 27, 28, and 29, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise an amino acid sequence having at least 80% identity with the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. vii. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively. viii. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 33, and 32, respectively; or ix. Comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively.

[0010] However, the sequence of SEQ ID NO: 27 is GFNIX2DTYIH, and X2 is K or E. The sequence of SEQ ID NO: 34 is SASX1LYS, and X1 is F or Y.

[0011] In some embodiments, the first antigen-binding fragment comprises a heavy chain variable region of trastuzumab or a variant thereof and a light chain variable region of trastuzumab or a variant thereof, wherein the variant heavy chain variable region comprises a K30E mutation according to the Kabat numbering system and the variant light chain variable region comprises an F53Y mutation according to the Kabat numbering system.

[0012] In some embodiments, the first antigen-binding fragment is chosen from: i. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 41 and 42, respectively. ii. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 41 and 42, respectively. iii. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 35 and 36, respectively. iv. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 35 and 39, respectively; or v. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 40 and 36, respectively. However, the sequence of SEQ ID NO: 41 is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIX2DTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, where X2 is K or E. The sequence of SEQ ID NO: 42 is as follows: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASX1LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, where X1 is F or Y.

[0013] In some embodiments, the antigen-binding constructs described herein comprise a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells. In some embodiments, the second antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3 of pertuzumab. In some embodiments, the second antigen-binding fragment comprises a heavy chain variable region of pertuzumab and a light chain variable region of pertuzumab.

[0014] In one aspect of the present invention there is provided a pharmaceutical composition comprising an antigen-binding construct as described herein and a pharmaceutically acceptable carrier.

[0015] In one aspect of the invention, there is provided an isolated nucleic acid or an isolated collection of nucleic acids comprising at least one nucleic acid sequence encoding at least one antigen-binding fragment of an antigen-binding construct described herein.

[0016] In one aspect of the invention, there is provided a vector or vector collection comprising one or more of the nucleic acids or nucleic acid collections described herein.

[0017] In one aspect of the invention, there is provided an isolated cell comprising a nucleic acid or collection of nucleic acids described herein, or a vector or collection of vectors described herein.

[0018] In one aspect of the invention there is provided a method of producing an antigen-binding construct as described herein, comprising culturing a host cell under conditions suitable for expression of said antigen-binding construct, said host cell comprising a nucleic acid encoding an antigen-binding construct as described herein, or a vector or vector collection as described herein, and purifying said construct.

[0019] In a further aspect of the invention, there is provided a method of treating a subject having a HER2-expressing tumor comprising administering to the subject a therapeutically effective amount of an antigen-binding construct described herein or a pharmaceutical composition described herein.

[0020] In a further aspect of the invention, there is provided a method of killing or inhibiting the growth of HER2-expressing tumor cells comprising contacting the tumor cells with an antigen-binding construct described herein.

[0021] In a further aspect of the invention, there is provided a method of inhibiting, reducing or blocking HER2 signalling in a cell comprising administering to the cell an effective amount of an antigen-binding construct as described herein or a pharmaceutical composition as described herein.

[0022] In a further aspect of the invention, there is provided a method of detecting or measuring HER2 in a sample comprising contacting the sample with an antigen-binding construct as described herein and detecting or measuring the bound complex.

[0023] In a further aspect of the present invention, there is provided a method for enhancing the aggregation resistance of an antigen-binding construct, the antigen-binding construct comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, the first antigen-binding fragment being an scFv, the method comprising modifying the antigen-binding construct so that the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 of the Kabat numbering system, where the mutated amino acid is E, and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 of the Kabat numbering system, where the mutated amino acid is Y. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows the glycan analysis of the anti-HER2 bispecific antibody Expi Her2-2. [Figure 2] FIG. 2 shows the glycan analysis of the anti-HER2 bispecific antibody 23C2 Her2-2. [Figure 3] Figure 3 shows the mortality of BT474 tumor cells with the combination of trastuzumab and pertuzumab, and with an anti-HER2 bispecific antibody. [Figure 4] Figure 4 shows the killing rates of trastuzumab, the combination of trastuzumab and pertuzumab, T-DM1, and an anti-HER2 bispecific antibody against NCI-N87 tumor cells. [Figure 5] Figure 5 shows the killing rates of trastuzumab, the combination of trastuzumab and pertuzumab, T-DM1, and an anti-HER2 bispecific antibody against JIMT-1 tumor cells. [Figure 6] FIG. 6 shows the growth inhibition of BT474 tumor cells by trastuzumab, a combination of trastuzumab and pertuzumab, and an anti-HER2 bispecific antibody. [Figure 7]Figure 7 shows the effect of a combination of trastuzumab and pertuzumab, and an anti-HER2 bispecific antibody on tumor volume changes in mice in an N87 gastric cancer xenograft mouse model. [Figure 8] Figure 8 shows the effect of a combination of trastuzumab and pertuzumab, and an anti-HER2 bispecific antibody on mouse weight change in response to gastric cancer N87 mouse xenograft tumors. [Figure 9] Figure 9 shows the structures of some exemplary anti-HER2 bispecific antibodies. One black chain (first Fc polypeptide) and one gray chain (second Fc polypeptide) represent a dimeric Fc, where the first antigen-binding fragment is an scFv fused to the first Fc polypeptide and the second antigen-binding fragment is a Fab fused to the second Fc polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0025] "term" The term "antigen-binding construct" refers to any substance capable of binding to an antigen, e.g., a polypeptide or polypeptide complex. In some embodiments, the antigen-binding construct is a polypeptide that specifically binds to a target antigen. The antigen-binding construct may be a monomer, dimer, multimer, protein, peptide, protein, peptide complex, antibody or antigen-binding portion thereof, scFv, etc. The antigen-binding construct may be a monospecific, bispecific, or multispecific polypeptide construct. In some embodiments, the antigen-binding construct comprises, for example, one or more antigen-binding moieties (e.g., Fab or scFv) connected to one or more Fc. Other examples of antigen-binding constructs are provided in the following description of the examples.

[0026] The term "bispecific" refers to any substance, such as an antigen-binding construct, having two antigen-binding portions (e.g., antigen-binding fragments), each with its own binding specificity. For example, a first antigen-binding fragment binds to an epitope on a first antigen, and a second antigen-binding fragment binds to an epitope on a second antigen. Alternatively, for example, the first and second antigen-binding fragments each bind to different epitopes on the same antigen.

[0027] The term "antibody" is used in the broadest sense to include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), multifunctional antibodies, and antibody fragments, provided they possess the desired biological activity.

[0028] The term "variable region" refers to the domain of approximately 100 to 110 or more amino acids located in the N-terminal domain of the light or heavy chain of an antibody, which is primarily responsible for antigen recognition. The terms "light chain variable region (VL)" and "heavy chain variable region (VH)" refer to these light chain and heavy chain domains, respectively.

[0029] The term "CDR (complementarity determining region)" is synonymous with "hypervariable region" and refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Natural tetrabodies generally contain six CDRs, three located in the heavy chain variable region and three in the light chain variable region.

[0030] The term "Fab" refers to a molecule containing a light chain constant domain (CL) and a heavy chain first constant domain (CH1), as well as variable domains VL (light chain variable region) and VH (heavy chain variable region) located in the light and heavy chains, respectively. The variable domains contain the complementarity-determining regions (CDRs) involved in antigen binding.

[0031] The term "scFv" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the scFv further comprises a polypeptide linker between the VH and VL domains that enables it to form the structure required for antigen binding.

[0032] The term "ECD" refers to the extracellular domain. The extracellular domain of HER2 contains four domains: ECD1, ECD2, ECD3, and ECD4.

[0033] Trastuzumab (generic name: Trastuzumab) is a recombinant humanized monoclonal antibody that selectively acts on the ECD4 of human epidermal growth factor receptor-2 (HER2) and can be used to treat HER2-positive cancers. An example of this is the therapeutic monoclonal antibody product sold under the trade name HERCEPTIN®.

[0034] Pertuzumab (generic name: Pertuzumab) is a recombinant humanized monoclonal antibody that selectively acts on the ECD2 of human epidermal growth factor receptor-2 (HER2) and can be used to treat HER2-positive cancers.

[0035] The term "HER2" refers to the second member of the EGFR family, which has tyrosine kinase activity.

[0036] The term "operably linked" refers to a functional connection between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of proteins, antibodies, or other polypeptides, the term "operably linked" refers to the joining of two or more amino acid segments in such a way that a functional polypeptide results.

[0037] The term “EC 50 " refers to the effective concentration of an antigen-binding construct that elicits a 50% maximal response. As used herein, the term "IC 50 " refers to the inhibitory concentration that elicits 50% of the maximal response of an antigen-binding construct. EC50 and IC 50 Both can be measured by ELISA, FACS analysis or any other method known in the art.

[0038] As used herein, the term "K D " is the equilibrium dissociation constant in molar concentration (M). K D The K value can be measured by methods well known in the art. D As a measurement method, surface plasmon resonance is preferred, and a biosensor system is more preferred. For example, Bia It is a core system.

[0039] The term "treatment" refers to a procedure used to treat, cure, alleviate, relieve, alter, repair, improve, ameliorate, or affect a disease (e.g., a disorder), symptoms of a disease, or prevent or delay the appearance of symptoms, complications, or biochemical indicators, or otherwise slow or inhibit the progression of a disease, disorder, or condition in a statistically significant manner.

[0040] A "therapeutically effective amount" refers to the amount of an antigen-binding construct or composition required to provide a therapeutic and / or prophylactic benefit to a subject.

[0041] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, both mammalian and non-mammalian, including non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, the subject of the present invention is a human. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably.

[0042] As used herein, "about" refers to an acceptable range of error for a particular value, determined by a person skilled in the art, and is partially determined by the limitations of the measurement method, i.e., the measurement system. For example, according to common knowledge in the art, "about" refers to a range of 1 or more standard deviations. Alternatively, "about" refers to a range of up to 5% (i.e., ±5%), and for example, the range of the indicated numerical value may vary within a range of ±2%, ±1%, or ±0.5%. When a specific value is indicated in this specification or claims, unless otherwise specified, the meaning of "about" is understood to be within the acceptable error range for the specific value.

[0043] The term "identity" is synonymous with identity. The percent identity of two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100%), taking into account the number of gaps and the length of each gap to be introduced to achieve optimal alignment of the two sequences. As shown in the following non-limiting examples, mathematical algorithms can be used to compare sequences and determine the percent identity of two sequences. The algorithm by E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) can be used to determine the percent identity of two amino acid sequences, and this algorithm uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and is already incorporated into the ALIGN program (version 2.0). Alternatively, the percent identity of two amino acid sequences can be determined using the algorithm by Needleman and Wunsch (J. Mol. Biol. 484-453 (1970)), which uses a Blossum 62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, and is already incorporated into the GAP program in the GCG software package (available at www.gcg.com).

[0044] The terms "Xn" and "Xaa" are the same and refer to unspecified amino acids. When using this term, the range is specifically defined.

[0045] In this specification, unless otherwise stated, the term "comprises" or equivalents (e.g., having, comprising, etc.) is an open term meaning including, but not limited to, recited elements, steps or structures, and is open to unrecited elements, steps or structures.

[0046] In this specification, terms used in the singular refer to plural referents unless otherwise specified. The same is true in the opposite case.

[0047] For purposes of explanation and disclosure, patents, patent applications, or existing publications are incorporated by reference in their entirety herein. These publications are provided because they were made public prior to the filing date of the present application. Any statement regarding the disclosure date of such documents or representation of their contents is based on information known to the applicant and does not constitute an admission that the disclosure date of such documents or their contents are correct. Furthermore, reference to such publications herein does not constitute an admission that such publications constitute common general knowledge in the art in all applicable countries. The following sections describe each aspect of the present invention in detail.

[0048] "Antigen-binding constructs" The present invention provides an antigen-binding construct comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, wherein the first antigen-binding fragment is an scFv, wherein the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system (in some examples, the mutated amino acid at position 30 is E), and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system (in some examples, the mutated amino acid at position 53 is Y).

[0049] In some embodiments, the antigen-binding constructs herein further comprise a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells.

[0050] In some embodiments, the antigen-binding constructs described herein comprise an immunoglobulin domain, e.g., one or more of the CL region, CH1 region, CH2 region, and / or CH3 region of an antibody, operably linked to a first antigen-binding fragment or / and a second antigen-binding fragment.

[0051] In some embodiments, the antigen-binding constructs described herein comprise a constant region operably linked to the first antigen-binding fragment and / or the second antigen-binding fragment. The constant region may be a native or mutated immunoglobulin constant region, e.g., one or more native or mutated CL, CH1, CH2, and / or CH3 domains, and in some instances, these domains are operably linked according to methods known in the art. The constant region may be derived from a human immunoglobulin constant region, e.g., from IgG1, IgG2, IgG3, or IgG4. In some instances, the constant region may have modifications to improve its ability to mediate effector function.

[0052] In some embodiments, the antigen-binding constructs described herein comprise an immunoglobulin domain or skeleton, e.g., Fc, operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment. The term "Fc" includes native sequence Fc regions and variant Fc regions. The Fc may be human, e.g., derived from IgG1, IgG2, IgG3, or IgG4. The Fc may have modifications to improve its ability to mediate effector function; for example, in some embodiments, the skeleton has modifications such as knob-in-hole, H435R, Y436F, defucosylation, etc.

[0053] The antigen-binding constructs according to the present invention may be monovalent, bivalent or multivalent. They may be monospecific, bispecific or multispecific. In some embodiments, the antigen-binding constructs herein are antibodies.

[0054] The inventors have unexpectedly discovered that introducing mutations into the variable region of the first antigen-binding fragment in scFV format of the antigen-binding construct can reduce aggregation of the antigen-binding construct. These mutations may be, for example, a mutation of amino acid at position 30 (according to the Kabat numbering system) in the heavy chain variable region of the first antigen-binding fragment to a negatively charged glutamic acid (E), or a mutation of amino acid at position 53 (according to the Kabat numbering system) in the light chain variable region of the first antigen-binding fragment to an uncharged tyrosine (Y), or the simultaneous introduction of these two mutations. Furthermore, the inventors have discovered that when a bispecific antigen-binding construct is constructed directly from an unmutated first antigen-binding fragment in scFV format (i.e., not E at position 30 and / or not Y at position 53), the undesired aggregation properties are inherited by the bispecific antigen-binding construct, whereas bispecific antibodies constructed by introducing the mutations described herein into the scFV exhibit excellent anti-aggregation properties. In some embodiments, the affinity of an antigen-binding construct (e.g., a monospecific or bispecific antigen-binding construct) for the HER2 antigen is not significantly reduced even after the mutations are introduced. In some embodiments, the antigen-binding construct has good affinity for the HER2 antigen. In some embodiments, a bispecific antigen-binding construct (e.g., 23C2 Her2-2) exhibits higher binding affinity to the Her2 antigen than trastuzumab or pertuzumab. In some embodiments, the K of the antigen-binding construct for the Her2 antigen is D is about 1E-8M or less, about 1E-9M or less, about 6.11E-10M or less, about 1E-11M or less, or about 1E-11M or less. In some embodiments, the Her2 antigen is a human Her2 antigen. In some embodiments, the binding affinity K of the antigen-binding constructs described herein Dis measured by Biacore.

[0055] In some embodiments, the antigen-binding constructs herein exhibit excellent anti-tumor properties, such as tumor cell killing and tumor growth inhibition. In some embodiments, the antigen-binding constructs constructed herein exhibit good killing effects against various tumor cells, including cells with different levels of HER2 expression. For example, some antigen-binding constructs exhibit good killing effects against BT474 (Her2+++) tumor cells. Some antigen-binding constructs exhibit good killing effects against NCI-N87 (Her2++) tumor cells. Some antigen-binding constructs exhibit good killing effects against trastuzumab-resistant tumor cells (e.g., JIMT-1). Additionally, some antigen-binding constructs exhibit good killing effects against tumor cells with low Her2 expression (e.g., MCF-7 Her2 0 / +).

[0056] In some embodiments, mouse model experiments have been performed that show that the antigen-binding constructs herein have no apparent toxic effects.

[0057] "Bispecific antigen binding construct" Provided herein are bispecific antigen-binding constructs that bind to HER2. The bispecific antigen-binding constructs comprise two antigen-binding fragments, each of which binds to a specific domain or epitope of HER2.

[0058] The antigen-binding constructs described herein are bispecific antigen-binding constructs comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, and a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells. wherein the first antigen-binding fragment is an scFv, and the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, where the mutated amino acid is E, or / and the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system; The amino acid after the mutation is Y.

[0059] In some embodiments, the antigen-binding construct described herein is a bispecific antigen-binding construct, wherein the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, wherein the mutated amino acid is E.

[0060] In some embodiments, the antigen-binding construct described herein is a bispecific antigen-binding construct, wherein the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, wherein the mutated amino acid is Y.

[0061] In some embodiments, the antigen-binding construct described herein is a bispecific antigen-binding construct, wherein the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, where the mutated amino acid is E, and the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, where the mutated amino acid is Y.

[0062] In some embodiments, the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, where the amino acid before the mutation is not E. In some embodiments, the amino acid before the mutation is K, i.e., in some embodiments, the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system.

[0063] In some embodiments, the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, where the amino acid prior to the mutation is not Y. In some embodiments, the amino acid prior to the mutation is F, i.e., in some embodiments, the light chain variable region of the first antigen-binding fragment comprises an F53Y mutation according to the Kabat numbering system.

[0064] In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise an amino acid sequence having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO: 27, 28, and 29, respectively. In certain embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively.

[0065] In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, light chain CDR2, and light chain CDR3 have at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, or at least 91% identity to the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. In some specific embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively.

[0066] In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 have at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, or at least 96% identity to the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. , or at least 97% identity, at least 98% identity, or at least 99% identity, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise amino acid sequences having at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively.In some examples, the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, respectively, of SEQ ID NOs: 43, 28, 29, 30, 31, and 32. In some examples, the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, respectively, of SEQ ID NOs: 43, 28, 29, 30, 33, and 32. In some examples, the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, respectively, of SEQ ID NOs: 44, 28, 29, 30, 31, and 32. In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a variant of a heavy chain variable region of trastuzumab and a light chain variable region of trastuzumab, and the variant of the heavy chain variable region comprises a K30E mutation according to the Kabat numbering system.

[0067] In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, wherein the first antigen-binding fragment comprises a heavy chain variable region of trastuzumab and a variant of a light chain variable region of trastuzumab, wherein the variant light chain variable region comprises an F53Y mutation according to the Kabat numbering system.

[0068] In some embodiments, the antigen-binding constructs herein are bispecific antigen-binding constructs. wherein the first antigen-binding fragment comprises a variant heavy chain variable region of trastuzumab and a variant light chain variable region of trastuzumab, wherein the variant heavy chain variable region comprises a K30E mutation and the variant light chain variable region comprises a F53Y mutation.

[0069] The first antigen-binding fragment is an scFV comprising a heavy chain variable region and a light chain variable region, and in some embodiments, the heavy chain variable region is the heavy chain variable region of trastuzumab or a variant thereof, and the light chain variable region is the light chain variable region of trastuzumab or a variant thereof.

[0070] In some embodiments, the antigen-binding construct herein is a bispecific antigen-binding construct, and the heavy chain variable region and light chain variable region of the first antigen-binding fragment comprise amino acid sequences having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequences of SEQ ID NOs: 41 and 42, respectively. In some embodiments, the first antigen-binding fragment comprises any of the CDR region sequence features described herein. In some embodiments, the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region comprise the amino acid sequences of SEQ ID NOs: 41 and 42, respectively. In some embodiments, the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and light chain variable region comprise the amino acid sequences of SEQ ID NOs: 35 and 36, respectively. In some embodiments, the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and light chain variable region comprise the amino acid sequences of SEQ ID NOs: 35 and 39, respectively. In some embodiments, the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and light chain variable region comprise the amino acid sequences of SEQ ID NOs: 40 and 36, respectively.

[0071] In some embodiments, the heavy chain variable region and light chain variable region of the first antigen-binding fragment are connected by a linker to form an scFV. The linker may be any suitable linker, for example, a charged and / or flexible linker. In certain embodiments, the linker consists of 1 to 50 amino acids connected by peptide bonds, and the amino acids may be selected from the 20 naturally occurring amino acids. In a more preferred embodiment, the 1 to 50 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, the linker consists essentially of amino acids that are not sterically hindered (e.g., glycine, alanine). A particularly preferred flexible linker is (GGGGS). n ((G4S) n (Also referred to as "flexible linker"). In some embodiments, n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any range limited by any two of the foregoing numbers, e.g., 1-5, 2-5, 3-6, 2-4, 1-4, etc. In some specific embodiments, the linker comprises a flexible connecting peptide having the sequence GGSGGSGGSGGSGG (SEQ ID NO: 52). In some specific embodiments, the linker comprises a flexible connecting peptide having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 53). In some specific embodiments, the linker comprises a flexible connecting peptide having the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 54). In some specific embodiments, the linker comprises a flexible connecting peptide having the sequence GGGPGKR (SEQ ID NO: 55). Examples of other suitable linkers include those listed in Table 4 of patent application WO2019195535.

[0072] In some embodiments, the antigen-binding constructs described herein are bispecific antigen-binding In the construct, the VH and VL of the first antigen-binding fragment are arranged from N- to C-terminus in the order VH-linker-VL, hi some embodiments, the VH and VL of the first antigen-binding fragment are arranged from N- to C-terminus in the order VL-linker-VH.

[0073] In some embodiments, the antigen-binding construct described herein is a bispecific antigen-binding construct, and the second antigen-binding fragment is a Fab. The second antigen-binding fragment specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells.

[0074] In some embodiments, the second antigen-binding fragment comprises the heavy chain CDR1, CDR2, and CDR3 of pertuzumab. In some embodiments, the second antigen-binding fragment comprises the light chain CDR1, CDR2, and CDR3 of pertuzumab. In some embodiments, the second antigen-binding fragment comprises the heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2, and CDR3 of pertuzumab. The amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of pertuzumab are GFTFTDYTMD (sequence number 45), DVNPNSGGSIYNQRFKG (sequence number 46), NLGPSFYFDY (sequence number 47), KASQDVSIGVA (sequence number 48), SASYRYT (sequence number 49), and QQYYIYPYT (sequence number 50), respectively.

[0075] In some embodiments, the second antigen-binding fragment comprises the heavy chain variable region and light chain variable region of pertuzumab. In some embodiments, the second antigen-binding fragment is a Fab fragment of pertuzumab. The amino acid sequence of the heavy chain variable region of pertuzumab is set forth in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region of pertuzumab is set forth in SEQ ID NO: 38.

[0076] In some embodiments, the antigen-binding constructs described herein are bispecific antigen-binding constructs comprising a constant region. In some embodiments, the constant region is connected to the first antigen-binding fragment and the second antigen-binding fragment via a linker. In some embodiments, the constant region is directly connected to the first antigen-binding fragment and the second antigen-binding fragment.

[0077] In some embodiments, the antigen-binding constructs described herein are bispecific antigen-binding constructs comprising a skeleton. In some embodiments, the skeleton is connected to the first antigen-binding fragment and the second antigen-binding fragment via a linker polypeptide (e.g., an immunoglobulin hinge region polypeptide selected from an IgG1, IgG2, or IgG4 hinge region). In some embodiments, the skeleton is directly connected to the first antigen-binding fragment and the second antigen-binding fragment.

[0078] In some embodiments, the scaffold is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide. As used herein, Fc is defined as the C-terminal region of at least a portion of the constant region, including an immunoglobulin heavy chain. The Fc polypeptide of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide capable of stably self-associating and including the C-terminal constant region of an immunoglobulin heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region conforms to the EU numbering system. For example, the Fc polypeptide of a dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences. The CH3 domain comprises two CH3 sequences derived from the first Fc polypeptide and the second Fc polypeptide, respectively, and the CH2 domain comprises two CH2 sequences derived from the first Fc polypeptide and the second Fc polypeptide, respectively. In some embodiments, the first antigen-binding fragment comprises the first Fc polypeptide. The first antigen-binding fragment is operably connected to a first Fc polypeptide, and the second antigen-binding fragment is operably connected to a second Fc polypeptide. In some embodiments, the first antigen-binding fragment and the first Fc polypeptide are operably connected via a first polypeptide linker, and the second antigen-binding fragment and the second Fc polypeptide are operably connected via a second polypeptide linker. The first linker polypeptide and the second linker polypeptide can form a covalent bond, e.g., a disulfide bond, with each other. In some embodiments, the first linker polypeptide or / and the second linker polypeptide comprise an IgG1 hinge region. In some embodiments, the first linker polypeptide or / and the second linker polypeptide comprise an IgG2 hinge region. In some embodiments, the first linker polypeptide or / and the second linker polypeptide comprise an IgG3 hinge region. In some embodiments, the first linker polypeptide or / and the second linker polypeptide comprise an IgG4 hinge region.

[0079] In some embodiments, the dimeric Fc is a human IgG1 Fc. In some embodiments, the dimeric Fc is a human IgG4 Fc.

[0080] In some embodiments, the dimeric Fc comprises a modification. In some embodiments, the dimeric Fc comprises H435R and / or Y436F, and the modification may be located in either the first Fc polypeptide or the second Fc polypeptide. In some specific embodiments, the dimeric Fc comprises H435R and / or Y436F, and the modification is present in only one Fc polypeptide but not the other. In some embodiments, the dimeric Fc comprises a knob-into-hole mutation site such as Y349C, T366S, L368A, Y407V, S354C, or T366W. In some embodiments, one chain of the dimeric Fc comprises T366Y / W and / or S354C, and the other chain comprises Y407T / V, Y349C, T366S, or / and L368A.

[0081] In some embodiments, the dimeric Fc does not comprise fucose.

[0082] In some embodiments, the antigen-binding constructs herein are glycosylated.

[0083] In some embodiments, the antigen-binding constructs herein are defucosylated. Defucosylation can improve the interaction between IgG1 and FcgRIIIa, thereby enhancing the ADCC effect of the antibody. Methods for generating antigen-binding constructs with very little or no fucose at the Fc glycosylation site, without altering the amino acid sequence, are known in the art. For example, these methods include adjusting the components of the medium containing the expressing cells, or knocking out a fucose expression-related gene, such as FUT8, from the expressing cells.

[0084] In some embodiments, the bispecific antigen-binding constructs described herein are bivalent.

[0085] Table S1 provides examples of bispecific antigen-binding constructs. In some embodiments, the bispecific antigen-binding constructs herein are bispecific antibodies. Table S2 provides examples of bispecific antibodies, such as Expi Her2-2, Expi Her2-3, Expi Her2-4, 23C-HER2-2, 23C-HER2-3, and 23C-HER2-4. The sequences of the CDR and variable regions are provided in Table S2. Exemplary bispecific antibodies comprise two heavy chains and one light chain, and the amino acid sequences of the heavy chains of Expi Her2-2 and 23C-HER2-2 are set forth in SEQ ID NOs: 11 and 13, and the amino acid sequences of the light chains are set forth in SEQ ID NO: 15. The amino acid sequences of the heavy chains of Expi Her2-3 and 23C-HER2-3 are set forth in SEQ ID NOs: 21 and 13, and the amino acid sequences of the light chains are set forth in SEQ ID NO: 15. The amino acid sequences of the heavy chains of Expi Her2-4 and 23C-HER2-4 are shown in SEQ ID NO:23 and SEQ ID NO:13, respectively, and the amino acid sequences of the light chains are shown in SEQ ID NO:15.

[0086] Further provided herein are several bispecific antigen-binding constructs with enhanced ADCC. Provided herein are exemplary bispecific antibodies with enhanced ADCC: 23C-HER2-2, 23C-HER2-3, and 23C-HER2-4. [Table 1] [Table 2] TIFF2026004416000003.tif210170

[0087] "Monospecific antigen-binding constructs" A monospecific antigen-binding construct refers to an antigen-binding construct that has one binding specificity.

[0088] In some embodiments, the antigen-binding constructs described herein are monospecific antigen-binding constructs that are monovalent and specifically bind to the ECD4 antigen of HER2 in HER2-expressing cells. a first antigen-binding fragment comprising an scFv, wherein the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, where the mutated amino acid is E, and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, where the mutated amino acid is Y.

[0089] In some embodiments, the antigen-binding constructs described herein are bivalent monospecific antigen-binding constructs that comprise two of the first antigen-binding fragments that are monovalent and specifically bind to the ECD4 antigen of HER2 in HER2-expressing cells.

[0090] In some embodiments, the antigen-binding constructs described herein are monospecific antigen-binding constructs, the first antigen-binding fragment of which may have a sequence characteristic of the first antigen-binding fragment of the bispecific antigen-binding constructs described herein.

[0091] In some embodiments, the antigen-binding constructs described herein are bivalent, monospecific antigen-binding constructs and comprise an Fc operably linked to a first antigen-binding fragment. In some embodiments, the Fc is a human IgG1 Fc.

[0092] In some embodiments, the antigen-binding construct is a bivalent monospecific antibody comprising the amino acid sequence of SEQ ID NO:3.

[0093] In some embodiments, the antigen-binding construct is a bivalent, monospecific antibody comprising the amino acid sequence of SEQ ID NO:9.

[0094] In some embodiments, the antigen-binding construct is a bivalent monospecific antibody comprising the amino acid sequence of SEQ ID NO:51.

[0095] "composition" Provided herein are pharmaceutical compositions comprising an antigen-binding construct or a nucleic acid encoding the antigen-binding construct, and further comprising one or more pharmaceutically acceptable carriers. In some embodiments, the composition comprises one or more bispecific antibodies, e.g., Expi Her2-2, Expi Her2-3, Expi Her2-4, 23C-HER2-2, 23C-HER2-3, or 23C-HER2-4, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0096] In the composition, the antigen-binding construct may be fully defucosylated (containing no detectable fucose) or partially defucosylated. In some embodiments, about 20% or less, about 18% or less, about 16% or less, about 14% or less, about 12% or less, about 10% or less, about 8% or less, about 6% or less, about 5% or less, about 3% or less, about 2% or less, about 1.5% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, or about 0% of the antigen-binding construct does not contain fucose. In some embodiments, about 0 to about 20%, about 0 to about 18%, about 0 to about 16%, about 0 to about 14%, about 0 to about 12%, about 0 to about 10%, about 0 to about 8%, about 0 to about 6%, about 0 to about 5%, about 0 to about 3%, about 0 to about 2%, about 0 to about 1.5%, about 0 to about 1%, about 0 to about 0.9%, about 0 to about 0.8%, about 0 to about 0.7%, about 0 to about 0.6%, about 0 to about 0.5%, about 0 to about 0.4%, about 0 to about 0.3%, about 0 to about 0.2%, about 0 to about 0.1%, or about 0.5% to approximately 0.6% does not contain fucose.

[0097] "Isolated nucleic acids" Provided herein is an isolated nucleic acid or an isolated collection of nucleic acids comprising at least one nucleic acid sequence encoding at least one polypeptide of an antigen-binding construct described herein. In some embodiments, the nucleic acid encodes a sequence of an antigen-binding construct described herein. In some embodiments, the nucleic acid can encode, for example, the amino acid sequence of a first antigen-binding fragment or / and a second antigen-binding fragment of an antigen-binding construct described herein. In some embodiments, the nucleic acid can encode, for example, the amino acid sequence of a heavy chain of an antigen-binding construct or / and a light chain of an antigen-binding construct described herein. The sequence listing includes exemplary nucleic acid sequences of several antigen-binding constructs, e.g., antigen-binding fragments of bispecific antibodies, and heavy and light chains of antigen-binding constructs.

[0098] "vector" The present invention provides a vector or collection of vectors comprising one or more of the isolated nucleic acids or collections of nucleic acids. In some embodiments, the vector is a cloning vector. In some embodiments, the vector is an expression vector.

[0099] Optionally, the expression vector is any expression vector capable of expressing an antigen-binding construct described herein.

[0100] "host cell" In some embodiments of the present invention, a host cell containing the vector is provided. The host cell is a suitable host cell for cloning or encoding an antigen-binding construct. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antigen-binding constructs. Mammalian cells include, for example, Chinese hamster ovary (CHO) cells and CHO-S cells.

[0101] Methods for producing antigen-binding constructs In some embodiments of the present invention, a method for producing an antigen-binding construct is provided, comprising culturing a host cell containing nucleic acid encoding the antigen-binding construct under conditions suitable for expression of the antigen-binding construct, and recovering the antigen-binding construct from the host cell or host cell culture medium. To produce the antigen-binding construct, the nucleic acid encoding the antigen-binding construct is isolated and inserted into one or more vectors for cloning and / or expression in the host cell. The nucleic acid can be obtained by a variety of methods known in the art, including gene splicing, chemical synthesis, and cloning.

[0102] "Usage" The present specification provides uses of antigen-binding constructs. In certain embodiments, the antigen-binding constructs used may be Expi Her2-2, Expi Her2-3, Expi Her2-4, 23C-HER2-2, 23C-HER2-3, or 23C-HER2-4.

[0103] In some embodiments, administering a therapeutically effective amount of an antigen-binding construct of the invention to a subject can kill HER2-expressing tumor cells or inhibit the growth of HER2-expressing tumor cells. In some embodiments of the invention, there is provided use of the antigen-binding construct in the manufacture of a medicament for killing HER2-expressing tumor cells or inhibiting the growth of HER2-expressing tumor cells. In some embodiments of the invention, there is provided use of the antigen-binding construct in the manufacture of a medicament for killing HER2-expressing tumor cells or inhibiting the growth of HER2-expressing tumor cells. Antigen-binding constructs for inhibiting the growth of HER2-expressing tumor cells are provided. In some embodiments of the present invention, therapeutic agents for killing HER2-expressing tumor cells or inhibiting the growth of HER2-expressing tumor cells are provided, comprising as an active ingredient an antigen-binding construct described herein.

[0104] In some embodiments, administering a therapeutically effective amount of an antigen-binding construct of the present invention to a subject can treat HER2-expressing tumors. Subjects in need of treatment include subjects who already have the disease or are in a state of the disease, subjects at risk of developing the disease or state, and subjects who may develop the disease or state and for whom the purpose is to prevent, delay, or alleviate the disease or state. In some embodiments of the present invention, use of the antigen-binding construct in the manufacture of a medicament for treating a subject with a HER2-expressing tumor is provided. In some embodiments of the present invention, an antigen-binding construct for treating HER2-expressing tumors is provided. In some embodiments of the present invention, a therapeutic agent for treating HER2-expressing tumors is provided, comprising as an active ingredient an antigen-binding construct described herein. Examples of tumors include, but are not limited to, biliary tract cancer, carcinosarcoma, esophageal cancer, esophagogastric junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer, and skin cancer.

[0105] In some embodiments, the tumor is HER2 0-1+, 1+, HER2 2+, or HER2 3+ as measured by IHC. In some embodiments, the tumor is HER2 2+ or less, or HER2 1+ or less.

[0106] In some embodiments, administering an effective amount of the antigen-binding construct to a cell can inhibit, reduce, or block HER2 signaling in the cell. In some embodiments of the present invention, there is provided use of the antigen-binding construct in the manufacture of a medicament for inhibiting, reducing, or blocking HER2 signaling in a cell. In some embodiments of the present invention, there is provided an antigen-binding construct for inhibiting, reducing, or blocking HER2 signaling in a cell. In some embodiments of the present invention, there is provided a therapeutic agent comprising as an active ingredient an antigen-binding construct described herein for inhibiting, reducing, or blocking HER2 signaling in a cell. In some embodiments, the cell is a tumor cell.

[0107] In some embodiments, there is provided a method of detecting or measuring HER2 in a sample comprising contacting the sample with an antigen-binding construct described herein and detecting or measuring the bound complex.

[0108] "Method for enhancing aggregation resistance of antigen-binding constructs" Provided herein is a method for enhancing the aggregation resistance of an antigen-binding construct, the antigen-binding construct comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, the first antigen-binding fragment being an scFv. The method comprises modifying the antigen-binding construct so that the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 of the Kabat numbering system, where the mutated amino acid is E, and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 of the Kabat numbering system, where the mutated amino acid is Y.

[0109] In some embodiments, the antigen-binding construct described herein further comprises a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells, wherein the second antigen-binding fragment is a Fab. In some embodiments, the first antigen-binding fragment comprises a group of CDRs selected from the following: i. It comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. ii. It comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. iii. It comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. iv. It comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. v. Comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. vi. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. vii. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively. viii. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 33, and 32, respectively; or ix. Comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively.

[0110] In some embodiments, the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, where the amino acid before the mutation is not E. In some embodiments, the amino acid before the mutation is K, i.e., in some embodiments, the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system.

[0111] In some embodiments, the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, where the amino acid prior to the mutation is not Y. In some embodiments, the amino acid prior to the mutation is F, i.e., in some embodiments, the light chain variable region of the first antigen-binding fragment comprises an F53Y mutation according to the Kabat numbering system.

[0112] In some embodiments, the antigen-binding construct is modified such that the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system and the light chain variable region of the first antigen-binding fragment comprises a F53Y mutation according to the Kabat numbering system. and comprising a heavy chain variable region of trastuzumab or a variant thereof and a light chain variable region of trastuzumab or a variant thereof, wherein the variant of the heavy chain variable region comprises a K30E mutation and the variant of the light chain variable region comprises an F53Y mutation.

[0113] In some embodiments, the method further comprises confirming the ability of the modified antigen-binding construct to bind to the HER2 antigen. In some embodiments, the method further comprises selecting a modified antigen-binding construct that binds to the HER2 antigen. In some embodiments, the affinity (e.g., K D ) is higher than or equivalent to the unmodified antigen-binding construct.

[0114] In some embodiments, the method further comprises selecting an antigen-binding construct that has a reduced tendency to aggregate compared to the unmodified antigen-binding construct.

[0115] For ease of understanding, the present invention has been described in detail using examples and embodiments, but it will be apparent to those skilled in the art that, based on the teachings of the present invention, certain changes and modifications can be made to the present invention without departing from the spirit and scope of the appended claims. The following examples are provided for illustrative purposes, not for limiting purposes. Those skilled in the art will be able to distinguish various non-critical parameters, which can be changed or modified to achieve similar results.

[0116] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, which are well known in the art, as described in detail in the following references: T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman & Co., 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc.); Sambrook et al., Molecular Cloning: A Laboratory Manual (1989, 2nd edition); Methods In Enzymology (S. Colowick, N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey, Sundberg Advanced Organic Chemistry 3rd edition (Plenum Press) volumes A and B (1992).

[0117] The present invention further provides the following embodiments, but the scope of protection of the present invention is not limited thereto. Embodiment 1: An antigen-binding construct comprising a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in HER2-expressing cells, wherein the first antigen-binding fragment is an scFv, and wherein the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, preferably where the mutated amino acid is E, and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, preferably where the mutated amino acid is Y.

[0118] Embodiment 2: The antigen-binding construct of embodiment 1, wherein the heavy chain variable region of the first antigen-binding fragment comprises a K30E mutation according to the Kabat numbering system, or / and the light chain variable region of the first antigen-binding fragment comprises a F53Y mutation according to the Kabat numbering system.

[0119] Embodiment 3: The antigen-binding construct of embodiment 1 or 2, which is an antibody.

[0120] Embodiment 4: The first antigen-binding fragment comprises a group of CDRs selected from: i. comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively; ii. comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively; iii. comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively; iv. comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively; v. comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively; vi. comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively; vii. comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively; viii. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 33, and 32, respectively; or ix. The antigen-binding construct of any one of embodiments 1 to 3, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively.

[0121] Embodiment 5: The antigen-binding construct of any one of embodiments 1 to 4, wherein the first antigen-binding fragment comprises a heavy chain variable region of trastuzumab or a variant thereof and a light chain variable region of trastuzumab or a variant thereof, wherein the variant heavy chain variable region comprises a K30E mutation according to the Kabat numbering system and the variant light chain variable region comprises a F53Y mutation according to the Kabat numbering system.

[0122] Embodiment 6: The first antigen-binding fragment is selected from: i. the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 41 and 42, respectively; ii. the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 41 and 42, respectively; iii. the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 35 and 36, respectively; iv. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 35 and 39, respectively; or v. The antigen-binding construct of embodiment 4 or 5, wherein the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 40 and 36, respectively.

[0123] Embodiment 7: The antigen-binding construct of any one of embodiments 1 to 6, wherein the VH and VL of the first antigen-binding fragment are arranged from N-terminus to C-terminus in the order VH-linker-VL.

[0124] Embodiment 8: The antigen-binding construct of any one of embodiments 1 to 7, further comprising a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells.

[0125] Embodiment 9: The antigen-binding construct of embodiment 8, wherein the second antigen-binding fragment is a Fab.

[0126] Embodiment 10: The antigen-binding construct of embodiment 8 or 9, wherein the second antigen-binding fragment comprises the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of pertuzumab.

[0127] Embodiment 11: The antigen-binding construct of embodiment 8 or 9, wherein said second antigen-binding fragment comprises the heavy chain variable region of pertuzumab and the light chain variable region of pertuzumab.

[0128] Embodiment 12: The antigen-binding construct of any one of embodiments 8 to 11, comprising an immunoglobulin domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, wherein the immunoglobulin domain comprises one or more of i.CL, CH1, CH2, CH3, or ii.Fc.

[0129] Embodiment 13: The antigen-binding construct of embodiment 12, wherein said CL, CH1, CH2, CH3, and Fc are derived from the CL, CH1, CH2, CH3, and Fc of human IgG, respectively.

[0130] Embodiment 14: The antigen-binding construct of embodiment 12, wherein the CL, CH1, CH2, CH3 or Fc has or does not have a modification, preferably wherein the modification in the CH3 or Fc is, for example, a substitution of the amino acid at position 435 or / and 436 according to the Kabat numbering system.

[0131] Embodiment 15: The antigen-binding construct of embodiment 12, wherein said Fc is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first antigen-binding fragment is operably linked to the first Fc polypeptide and the second antigen-binding fragment is operably linked to the second Fc polypeptide.

[0132] Embodiment 16: The antigen-binding construct of any one of embodiments 1 to 15, which is a bispecific or multispecific antibody.

[0133] Embodiment 17: The antigen-binding construct of any one of embodiments 1 to 15, which is bivalent or multivalent.

[0134] Embodiment 18: Any of embodiments 1 to 7, which is a monovalent, bivalent, or polyvalent monospecific antibody. 2. The antigen-binding construct of claim 1.

[0135] Embodiment 19: i. A bivalent, bispecific antibody comprising a heavy chain comprising SEQ ID NO: 11, a heavy chain comprising SEQ ID NO: 13, and a light chain comprising SEQ ID NO: 15; ii. a bivalent, bispecific antibody comprising a heavy chain comprising SEQ ID NO: 21, a heavy chain comprising SEQ ID NO: 13, and a light chain comprising SEQ ID NO: 15; iii. a bivalent, bispecific antibody comprising a heavy chain comprising SEQ ID NO: 23, a heavy chain comprising SEQ ID NO: 13, and a light chain comprising SEQ ID NO: 15; iv. a bivalent, monospecific antibody comprising the amino acid sequence of SEQ ID NO: 3; v. A bivalent, monospecific antibody comprising the amino acid sequence of SEQ ID NO: 9; and vi. a bivalent, monospecific antibody comprising the amino acid sequence of SEQ ID NO: 51.

[0136] Embodiment 20: has reduced aggregation compared to an antigen-binding construct in which position 30 is not E and / or position 53 is not Y, and / or 20. The antigen-binding construct of any one of embodiments 1 to 19, which has clearly not reduced binding affinity to the ECD4 antigen and / or ECD2 antigen of HER2 compared to an antigen-binding construct in which position 30 is not E and / or position 53 is not Y.

[0137] Embodiment 21: The antigen-binding construct of any one of embodiments 1 to 20, which is defucosylated.

[0138] Embodiment 22: A pharmaceutical composition comprising the antigen-binding construct of any one of embodiments 1 to 21 and a pharmaceutically acceptable carrier.

[0139] Embodiment 23: An isolated nucleic acid or an isolated collection of nucleic acids comprising at least one nucleic acid sequence encoding at least one antigen-binding fragment of the antigen-binding construct of any one of embodiments 1 to 21.

[0140] Embodiment 24: A vector or a collection of vectors comprising one or more of the nucleic acids or nucleic acid collections of embodiment 23.

[0141] Embodiment 25: An isolated cell comprising a nucleic acid or collection of nucleic acids according to embodiment 23, or a vector or collection of vectors according to embodiment 24.

[0142] Embodiment 26: A method for producing the antigen-binding construct of any one of Embodiments 1 to 21, comprising culturing host cells under conditions suitable for expression of the antigen-binding construct, wherein the host cells contain a nucleic acid encoding the antigen-binding construct, or a vector or vector collection of Embodiment 24, and purifying the construct.

[0143] Embodiment 27: A method for treating a subject having a HER2-expressing tumor, comprising administering to the subject a therapeutically effective amount of an antigen-binding construct of any one of embodiments 1 to 21 or a pharmaceutical composition of embodiment 22.

[0144] Embodiment 28: The method of embodiment 27, comprising killing or inhibiting the growth of HER2-expressing tumor cells by contacting the tumor cells with said antigen-binding construct.

[0145] Embodiment 29: The tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, esophagogastric junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, intrauterine cancer The method of embodiment 27 or 28, wherein the cancer is membranous, uterine, uterine, melanoma, pharyngeal, oral, or skin cancer.

[0146] Embodiment 30: The method of any one of embodiments 27 to 29, comprising inhibiting, reducing or blocking HER2 signaling in a cell by administering to the cell an effective amount of said antigen-binding construct or said pharmaceutical composition.

[0147] Embodiment 31: A method of enhancing the aggregation resistance of an antigen-binding construct, wherein the antigen-binding construct is monovalent and comprises a first antigen-binding fragment that specifically binds to the ECD4 antigen of HER2 in a HER2-expressing cell, wherein the first antigen-binding fragment is an scFv; modifying the antigen-binding construct so that the heavy chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 30 according to the Kabat numbering system, where the mutated amino acid is E, and / or the light chain variable region of the first antigen-binding fragment comprises an amino acid mutation at position 53 according to the Kabat numbering system, where the mutated amino acid is Y.

[0148] Embodiment 32: The method of embodiment 31 for enhancing the aggregation resistance of an antigen-binding construct, wherein the antigen-binding construct further comprises a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in HER2-expressing cells, and wherein the second antigen-binding fragment is a Fab.

[0149] Embodiment 33: A method of enhancing aggregation resistance of an antigen-binding construct of embodiment 31 or 32, wherein said first antigen-binding fragment comprises a group of CDRs selected from: i. It comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. ii. It comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. iii. It comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. iv. It comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity with the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. v. Comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. vi. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise amino acid sequences having at least 80% identity to the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively. vii. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively. viii. heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are They comprise the amino acid sequences of SEQ ID NOs: 43, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 33, and 32, respectively. ix. A heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively; or ix. Comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively.

[0150] Embodiment 34: A method of enhancing aggregation resistance of an antigen-binding construct of embodiment 31 or 32, wherein the first antigen-binding fragment is an scFv and comprises a heavy chain variable region of trastuzumab or a variant thereof and a light chain variable region of trastuzumab or a variant thereof, wherein the variant of the heavy chain variable region comprises a K30E mutation and the variant of the light chain variable region comprises a F53Y mutation.

[0151] Example 1: Construction, expression, and purification of anti-Her2-scFv-Fc and its variants When constructing anti-Her2-scFv-Fc, human IgG1 was used as the Fc portion, and the anti-Her2 arm variable region sequence was based on that of trastuzumab (Herceptin®) monoclonal antibody. The light and heavy chain variable regions of trastuzumab (Herceptin®) monoclonal antibody were linked using a designed linker 1 ((GGGGS)3, SEQ ID NO: 53) to form anti-Her2-scFv-Fc (SEQ ID NO: 1). The amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0152] Furthermore, point mutations were introduced into the anti-Her2-scFv-Fc sequence to construct the following mutants. Anti-Her2-scFv-VL-F53Y-Fc (SEQ ID NO: 3): Derived from wild-type anti-Her2-scFv-Fc, the VL region contains F53Y. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCR ASQDVNTAVAWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0153] Anti-Her2-scFv-VL-F53A-Fc (SEQ ID NO: 5): Derived from wild-type anti-Her2-scFv-Fc, the VL region contains F53A. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASALYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0154] Anti-Her2-scFv-VL-F53R-Fc (SEQ ID NO: 7): Derived from wild-type anti-Her2-scFv-Fc, the VL region contains F53R. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASRLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0155] Anti-Her2-scFv-VH-K30E-Fc (SEQ ID NO: 9): Derived from wild-type anti-Her2-scFv-Fc, the VH domain contains K30E. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIEDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0156] The DNA sequences of anti-Her2-scFv-Fc and its variants (SEQ ID NOs: 2, 4, 6, 8, and 10) were synthesized and cloned into the pcDNA3.1 expression vector. The expression vectors for anti-Her2-scFv-Fc or its variants were transfected into ExpiCHO cells (CHO-S, Thermo) using the ExpiCHO™ Expression Kit (Thermo Fisher, Cat. No. A29133). The cells were cultured in ExpiCHO expression medium at 37°C in an 8% CO2 humidified atmosphere incubator on an orbital shaker platform rotating at 130 rpm. The culture supernatant was collected and protein purification was performed using Protein A magnetic beads (Genscript, Cat. No. L00273). The protein was analyzed by a UV-Vis spectrophotometer (NanoDrop lite, Thermo). Protein concentration was measured using a ELISA kit (Scientific). [Table 3]

[0157] Example 2: Verification of aggregates of anti-Her2-scFv-Fc and its mutants and detection of binding to human Her2 antigen The affinity of the test molecules to human Her2 protein was measured using the expressed and purified anti-Her2-scFv-Fc (anti-Her2-scFv-Fc) and its variants using a Biacore T200 (GE) analyzer. The experimental procedure was as follows: A fixed amount of anti-Her2-scFv-Fc (anti-Her2-scFv-Fc) or its variants was captured on a chip bound to anti-hIgG (anti-hIgG), and then antigen human HER2 protein (Sino Biological, catalog number: 10004-H08H) was applied to the surface of the chip. Binding and dissociation curves were obtained by detecting the reaction signal in real time using a Biacore T200. Buffers used in the experiment was Biacore universal buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P20 (GE, catalog number: BR-1000-54), pH 7.4). Anti-hIgG (derived from a human antibody capture kit, GE, catalog number 29-2346-00) was bound to the surface of a CM5 chip, and approximately 9000 RU of anti-Her2-scFv-Fc or its variants were captured. The signal intensity of the interaction between anti-Her2-scFv-Fc or its variants and different concentrations of human HER2 protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) was then measured. The flow rate of the flow cell was 50 μL / min, and the association time was 240 s, the dissociation time was 1400 s, and the baseline was stabilized after 60 s of regeneration with 3 M MgCl (GE). The results were calculated using the affinity and kinetics 1:1 binding model of the biacore evaluation software. The affinity of anti-Her2-scFv-Fc (anti-Her2-scFv-Fc) or its variants to the antigen human Her2 protein is shown in Table 2. Anti-Her2-scFv-Fc (K D =0.77 nM) compared to the mutant anti-Her2-scFv-VL-F53A-Fc (anti-Her2-scFv-VL-F53A-Fc) (K D = 1.26 nM) had a significant effect on the binding affinity to the antigen human Her2 protein. D The binding affinity of anti-Her2-scFv-Fc (K = 0.8 nM) to the antigen human Her2 protein was D = 0.77 nM), and the binding of the mutant anti-Her2-scFv-VH-K30E-Fc to the antigen human Her2 protein was K D=0.54 nM. It was found that the introduction of the F53Y and K30E mutations did not reduce the binding affinity to the antigen human Her2 protein.

[0158] Anti-Her2-scFv-Fc and its variants were separated using a gel filtration chromatography column. The components were eluted in order of decreasing molecular weight using a neutral pH buffer solution. The chromatography column was an ACQUITY UPLC Protein BEH SEC Column (200 Å, 1.7 μm, 4.6 × 300 mm), and the column temperature was 25°C. The mobile phase was 50 mmol / L phosphate buffer solution-200 mmol / L sodium chloride, pH 7.0. (2.33 g of sodium dihydrogen phosphate dihydrate, 12.53 g of disodium hydrogen phosphate dodecahydrate, and 11.69 g of sodium chloride were weighed, added to approximately 800 mL of ultrapure water, stirred to dissolve thoroughly, and then added ultrapure water to make a 1000 mL solution. The mixture was homogenized and filtered through a 0.22 μm filter membrane.) The sample was diluted to 10 mg / mL with the mobile phase to prepare the test solution. 2 μL of the solution was accurately injected into the liquid chromatograph (if the sample concentration was less than 10 mg / mL, the injection volume was adjusted to 20 μg of protein). Detection was performed at 280 nm. The flow rate was 0.30 mL / min, and isocratic elution was performed for 15 minutes. Data were processed and quantitatively analyzed using the area percentage method. The peak area percentages of aggregates, immunoglobulin monomers, and low-molecular-weight impurities were calculated, with the area before the main peak representing aggregates, the main peak representing immunoglobulin monomers, and the area after the main peak representing low-molecular-weight impurities. The ratio of the main peak to the aggregate content of anti-Her2-scFv-Fc (anti-Her2-scFv-Fc) or its variants is shown in Table 2. The mutant anti-Her2-scFv-VL-F53Y-Fc and anti-Her2-scFv-VH-K30E-Fc significantly reduced aggregation, from 6.31% for anti-Her2-scFv-Fc to 4.94% and 3.39%, respectively. [Table 4]

[0159] Example 3: Construction, expression and purification of anti-Her2 bispecific antibodies An anti-Her2 bispecific antibody was generated as a human IgG1 antibody by modifying the Fc domain using the knobs-into-holes (Ridgway, et al., 1996) process. The H435R and Y436F mutations (Jendeberg et al., 1997) were engineered into the Fc domain sequence of one of the heavy chains to reduce the affinity of the Fc domain for Protein A. These mutations are useful for removing homodimers formed during the assembly of the bispecific antibody during Protein A affinity purification (Patent US5945311A). Example 2 shows that the anti-Her2-scFv-VL-F53Y-Fc mutation and the anti-Her2-scFv-VH-K30E-Fc mutation can clearly reduce anti-Her2-scFv aggregation without reducing the affinity for the Her2 antigen. In this example, the antigen-binding domain of one of the anti-Her2 arms of the anti-Her2 bispecific antibody is a scFv (VH- In the form of a linker-VL structure, the variable region sequences contained the mutation K30E (b-anti-Her2-scFv-VH-K30E-Fc, SEQ ID NO: 21), the mutation F53Y (b-anti-Her2-scFv-VL-F53Y-Fc, SEQ ID NO: 23), or both point mutations simultaneously, resulting in anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO: 11). In this example, the antigen-binding domain of the other anti-Her2 arm of the anti-Her2 bispecific antibody is in the form of Fab containing anti-Her2-domain2-HC-Fc (anti-Her2-domain2-HC-Fc) (SEQ ID NO: 13) and anti-Her2-domain2-LC (anti-Her2-domain2-LC) (SEQ ID NO: 15). In addition, a control anti-Her2 bispecific antibody was constructed using a mutation-free scFv (VH-linker-VL structure or VL-linker-VH structure). [Table 5] TIFF2026004416000007.tif209170 TIFF2026004416000008.tif194170 TIFF2026004416000009.tif225170 TIFF2026004416000010.tif65170

[0160] The DNA sequences of anti-Her2 bispecific antibodies (SEQ ID NOs: 12, 14, and 16) were synthesized and cloned into the pcDNA3.1 expression vector. Using the ExpiCHO™ Expression Kit (Thermo Fisher, catalog number: A29133), the expression vectors for anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (anti-Her2-scFv-VH-K30E-VL-F53Y-Fc) (SEQ ID NO: 11), anti-Her2-domain2-HC-Fc (anti-Her2-domain2-HC-Fc) (SEQ ID NO: 13), and anti-Her2-domain2-LC (anti-Her2-domain2-LC) (SEQ ID NO: 15) were co-transfected into ExpiCHO cells at a transfection ratio of 1:1:1.5. The transfection density was 6 × 10. 6 The cell concentration was 1000 cells / mL. The medium was ExpiCHO expression medium. The cells were continuously cultured for 10 days after transfection, and the cell culture supernatant was collected by centrifugation. Protein purification was performed using protein A magnetic beads (Genscript, catalog number: L00273). Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). The sample was named Expi Her2-2. Following this method, other bispecific antibodies, Expi Her2-1, Expi Her2-3, Expi Her2-4, and Expi Her2-5, were obtained by expression and purification using the DNA sequences shown in the table above.

[0161] Example 4: Production and validation of fucose knockout bispecific antibodies Knocking out the fucose expression-related gene FUT8 can improve the interaction between IgG1 and FcgRIIIa, thereby enhancing the ADCC effect of antibodies (Shields et al., 2002; Yamane-Ohnuki et al., 2004). In this example, fucose knockout anti-Her2 bispecific antibodies were produced in FUT8-knockout CHO-S cells (designated CHO FUT8- / - cells). The DNA sequences of the anti-Her2 bispecific antibodies (SEQ ID NOs: 12, 14, and 16) were synthesized and cloned into the pcDNA3.1 expression vector. FUT8-knockout CHO-S cells were co-transfected with expression vectors for anti-Her2-scFv-VH-K30E-VL-F53Y-Fc, anti-Her2-domain2-HC-Fc, and anti-Her2-domain2-LC at a transfection ratio of 1:1:1.5 using the CHOgro® High Yield Expression System (Cat. No. MIR 6270). The transfection density was 6 × 10 6 The cell density was 100 cells / mL. The medium was CHOgro® Expression Medium (Catalog No.: MIR 6200, Mirus). The cells were further cultured and centrifuged to collect the cell culture supernatant. Protein purification was performed using Protein A magnetic beads (Genscript, catalog number: L00273). Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). The sample was named 23C2 Her2-2. According to this method, the sequences of Expi Her2-1, Expi Her2-3, Expi Her2-4, and Expi Her2-5 shown in Table 3 were expressed in CHO FUT8- / - cells to obtain the fucose knockout anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5.

[0162] Anti-Her2 bispecific antibody samples expressed by CHO FUT8- / - cells and CHO-S cells were treated with the GlycoWorks RapiFluor-MS N-glycan kit (Waters, Milford, MA, USA) to release N-glycans from the proteins, which were then labeled and separated on a chromatography column. The N-glycan structure and content were then analyzed using an FLR detector (Waters, Milford, MA, USA). The glycan analysis results for the anti-Her2 bispecific antibody Expi HER2-2 are shown in Figure 1. The glycan analysis results for the anti-Her2 bispecific antibody 23C2 HER2-2 expressed by FUT8 knockout CHO-S cells are shown in Figure 2. The glycan content ratios are shown in Table 4. The results for the normal anti-Her2 bispecific antibody Expi HER2-2 are shown in Figure 1. The fucose-free ratio for HER2-2 was 21.85%, and the fucose-free ratio for expression of anti-Her2 bispecific antibody 23C2 HER2-2 in FUT8-knockout CHO-S cells was 99.40%. [Table 6] TIFF2026004416000012.tif140170

[0163] Example 5: Verification of bispecific antibody aggregates In this example, aggregates of anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5 were examined.

[0164] Anti-Her2 bispecific antibodies were separated using a gel filtration chromatography column and their aggregate content was verified. Elution was performed using a neutral pH buffer solution, with molecular weight components eluted in decreasing order. The column temperature was 25°C. The mobile phase was a 50 mmol / L phosphate buffer solution containing 200 mmol / L sodium chloride, pH 7.0. (2.33 g of sodium dihydrogen phosphate dihydrate, 12.53 g of disodium hydrogen phosphate dodecahydrate, and 11.69 g of sodium chloride were weighed, added to approximately 800 mL of ultrapure water, stirred to dissolve thoroughly, and then added ultrapure water to make a 1000 mL volume. The mixture was homogenized and filtered through a 0.22 μm filter membrane.) The sample was diluted to 10 mg / mL with the mobile phase to prepare a test solution, and 2 μL of the solution was precisely measured and injected into the liquid chromatograph (when the sample concentration was less than 10 mg / mL, the injection volume was adjusted to inject 20 μg of protein). Detection was performed at a wavelength of 280 nm. The flow rate was 0.30 mL / m. The elution was performed isocratically for 15 minutes at 1000 kJ / min. The data were processed and quantitatively analyzed using the area percentage method. The peak area percentages of aggregates, immunoglobulin monomers, and low-molecular-weight impurities were calculated, of which the area before the main peak was aggregates, the main peak was immunoglobulin monomers, and the area after the main peak was low-molecular-weight impurities. [Table 7]

[0165] As can be seen from the results, when the bispecific antibody was assembled from scFV (without mutations), the bispecific antibody still produced a large amount of aggregates, and the introduction of mutations reduced the aggregate content of the bispecific antibody, with the aggregate content of 23C2 Her2-2 being reduced to 8.51% compared to 23C2 Her2-1 without mutations (Table 5).

[0166] Example 6: Antigen binding detection of anti-Her2 bispecific antibodies The affinity of the test molecule to the HER2 protein of the expressed and purified anti-Her2 bispecific antibody was measured using Biacore T200 (GE), and the experimental procedure was as follows: A fixed amount of anti-Her2 bispecific antibody was captured on a chip pre-loaded with anti-hIgG. Human Her2 (Sino Biological, catalog number 10004-H08H) was then flowed over the chip surface, and binding and dissociation curves were obtained by detecting the reaction signal in real time using a Biacore T200. The buffer used in the experiment was Biacore universal buffer (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P20, pH 7.4). Anti-hIgG (derived from a human antibody capture kit, GE, catalog number 29-2346-00) was bound to the surface of a CM5 chip, resulting in a binding capacity of approximately 9000 RU. Approximately 200 RU of anti-Her2 bispecific antibody was then captured. The signal intensity of the interaction between the anti-Her2 bispecific antibody and Her2 protein at different concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) was then measured. The flow rate was 50 μL / min, and the association time was 240 s, followed by a dissociation time of 1400 s. The baseline was stabilized after 60 s of regeneration with 3 M MgCl2 (GE).

[0167] The results were calculated using biacore evaluation software. The binding affinity of the anti-Her2 bispecific antibody 23C2 Her2-2 to the antigen Her2 and the trastuzumab and pertuzumab controls are shown in Table 6. The K D is 6.11E-10 M, and the K D is 1.22E-09M, and the K D The affinity of the anti-Her2 bispecific antibody 23C2 Her2-2 for the antigen Her2 was 2.35E-09M, indicating that it had a higher affinity for the antigen Her2 than trastuzumab and pertuzumab. [Table 8]

[0168] Example 7: Antigen binding detection of anti-Her2 bispecific antibodies According to the experimental procedure of Example 6, the affinities of the expressed and purified anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5 to HER2 protein were measured using Biacore T200 (GE).

[0169] The affinity of anti-Her2 bispecific antibodies for the antigen, human Her2 protein, is shown in Table 7. The results in Tables 6 and 7 demonstrate that the introduction of the F53Y and K30E mutations can improve the binding affinity of anti-Her2 bispecific antibodies for the antigen, human Her2 protein. [Table 9]

[0170] Example 8: Killing of Her2-positive target cells BT474 by anti-Her2 bispecific antibodies We investigated the killing effect of anti-Her2 bispecific antibodies against target cells (BT474 Her2+++, provided by the Cell Bank of the Committee for the Collection of Typical Cultures of the Chinese Academy of Sciences) using NK cells derived from human PBMCs (peripheral blood mononuclear cells, effector cells). 50 was calculated to evaluate the in vitro activity of the anti-Her2 bispecific antibody.

[0171] The details of the experimental process are as follows: BT474 cells were cultured at a cell density of 3 × 10 in 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 The antibody was adjusted to 1.5 × 10 cells / mL and seeded into a 96-well cell culture plate (Eppendorf, catalog number: 0030730199) at 50 μL per well. Anti-Her2 bispecific antibodies were prepared at different concentrations (1000 ng / mL, 333 ng / mL, 111 ng / mL, 37 ng / mL, 12.3 ng / mL, 4.11 ng / mL, 1.37 ng / mL, 0.46 ng / mL, 0.15 ng / mL, and 0.05 ng / mL) using 1640 experimental medium. The antibody was added at different concentrations to the 96-well cell culture plate at 50 μL per well. Human PBMCs were seeded into a 96-well cell culture plate at a cell density of 1.5 × 10 cells / mL using 1640 experimental medium. 6 The concentration of cells / mL was adjusted to 100 μL per well. A target cell group (human PBMC), a target cell + effector cell group, a blank control group (medium), a lysate control group, and a target cell maximum release group (target cell + lysate) were set up, with an effector cell:target cell ratio of 10:1. Forty-five minutes before detection, 20 μL / well of lysate (Promega, catalog number: G182A) was added to the target cell maximum release group and the lysate control group. After 45 minutes, the cell lysis rate (death rate) was detected using the CytoTox96® nonradioactive cytotoxicity assay kit (Promega, G1780). Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 )×100%.

[0172] Figure 3 shows the killing rates of anti-Her2 bispecific antibodies against BT474 Her2+++ tumor cells. The ADCC-enhanced anti-Her2 bispecific antibodies 23C2 HER2-1 and 23C2 HER2-2 exceeded the killing rate of BT474 tumor cells by the combination of trastuzumab and pertuzumab, and also exceeded the killing rate of the anti-Her2 bispecific antibodies Expi Her2-1 and Expi HER2-2 expressed from CHO-S. The EC 50 was 8.627ng / mL, and the EC of Expi HER2-1 50 The EC was 38.05ng / mL, and Expi HER2-2 was superior to Expi HER2-1. 50 The EC of ADCC-enhanced 23C2 HER2-1 was 35.17ng / mL. 50 The EC was 4.728ng / mL, and the ADCC-enhanced 23C2 HER2-2 was superior to 23C2 HER2-1. 50 was 3.658ng / mL.

[0173] Example 9: Killing of Her2-positive target cells NCI-N87 by anti-Her2 bispecific antibodies We investigated the killing effect of anti-Her2 bispecific antibodies against target cells (NCI-N87 Her2++, provided by the Cell Bank of the Committee for the Collection of Typical Cultures of the Chinese Academy of Sciences) using NK cells derived from human PBMCs (peripheral blood mononuclear cells, effector cells). 50 was calculated to evaluate the in vitro activity of the anti-Her2 bispecific antibody.

[0174] The details of the experimental process are as follows: NCI-N87 cells were cultured at a cell density of 3 × 10 in 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 The cells / mL were adjusted and seeded into a 96-well cell culture plate (Eppendorf, catalog number: 0030730199) at 50 μL per well. Anti-Her2 bispecific antibodies or control drugs were prepared at different concentrations (8.1 nM, 2.7 nM, 0.9 nM, 0.3 nM, 0.1 nM, 0.03 nM, 0.01 nM, 0.003 nM, 0.001 nM, and 0.0004 nM) using 1640 experimental medium. The antibodies or control drugs at different concentrations were added to the 96-well cell culture plate at 50 μL per well. Human PBMCs were seeded into a 96-well cell culture plate at a cell density of 1.5 × 10 cells / mL using 1640 experimental medium. 6 The cells were adjusted to 100 μL per well. The treatment group (target cells + effector cells + antibody), target cell group (NCI-N87 cells), effector cell group (human PBMCs), target cell + effector cell group, blank control group (medium), lysate control group, and target cell maximum release group (target cells + lysate) were set up. The effector cell:target cell ratio was 10:1. Forty-five minutes before detection, 20 μL of lysate (Promega, catalog number G182A) was added to the target cell maximum release group and lysate control group. After 45 minutes, the cell lysis rate (death rate) was detected using the CytoTox96® nonradioactive cytotoxicity assay kit (Promega, G1780). Trastuzumab, T-DM1 (trastuzumab-maytansine conjugate, trade name Kadcyla The comparators were trastuzumab + pertuzumab (1:1), and Expi HER2-1. Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 )×100%.

[0175] Figure 4 shows the killing rates of NCI-N87 tumor cells by the combination of trastuzumab and pertuzumab, trastuzumab, T-DM1, and the anti-Her2 bispecific antibody. ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 was superior to trastuzumab + pertuzumab, trastuzumab, T-DM1, and Expi HER2-1 in killing NCI-N87 tumor cells. Among these, the ADCC-enhancing anti-Her2 (anti-Her2) bispecific antibody 23C2 HER2-2 had the highest EC 50 The EC of the combination of trastuzumab and pertuzumab was 0.02447 nM. 50 The EC of Expi HER2-1 was 0.08267nM. 50 The EC of T-DM1 was 0.1048 nM. 50 The EC value of trastuzumab was 0.07392 nM. 50 was 0.07468nM.

[0176] Example 10: Killing of trastuzumab-resistant JIMT-1 cells by anti-Her2 bispecific antibodies Using NK cells provided by human PBMCs (peripheral blood mononuclear cells, effector cells), we investigated the killing effect of anti-Her2 bispecific antibodies against target cells (JIMT-1, provided by AddexBio, catalog number: C0006005). 50 was calculated to evaluate the in vitro activity of the anti-Her2 bispecific antibody.

[0177] The details of the experiment process are as follows: JIMT-1 cells were cultured at a cell density of 3 × 10 in 1640 experimental medium containing 2% FBS (fetal bovine serum). 5The cells / mL were adjusted and seeded into a 96-well cell culture plate (Eppendorf, catalog number: 0030730199) at 50 μL per well. Anti-Her2 bispecific antibodies or control drugs were prepared at different concentrations (8.1 nM, 2.7 nM, 0.9 nM, 0.3 nM, 0.1 nM, 0.03 nM, 0.01 nM, 0.003 nM, 0.001 nM, and 0.0004 nM) using 1640 experimental medium. The antibodies or control drugs at different concentrations were added to the 96-well cell culture plate at 50 μL per well. Human PBMCs were seeded into a 96-well cell culture plate at a cell density of 1.5 × 10 cells / mL using 1640 experimental medium. 6 The cells were adjusted to 100 μL per well. Treatment groups (target cells + effector cells + antibody or control), target cells (BT474 cells), effector cells (human PBMCs), target cells + effector cells, blank control (medium), lysate control, and target cell maximum release group (target cells + lysate) were set up. The effector cell:target cell ratio was 20:1. Forty-five minutes before detection, 20 μL of lysate (Promega, catalog number G182A) was added to the target cell maximum release group and lysate control group. After 45 minutes, the cell lysis rate (death rate) was detected using the CytoTox96® nonradioactive cytotoxicity assay kit (Promega, G1780). The comparators were trastuzumab, T-DM1, trastuzumab + pertuzumab combination (1:1), and Expi HER2-1. Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 )×100%.

[0178] Figure 5 shows the killing rates of JIMT-1 tumor cells by the combination of trastuzumab and pertuzumab, trastuzumab, T-DM1, and the anti-Her2 bispecific antibody. The killing rate of JIMT-1 tumor cells by the ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 was significantly higher than that of the combination of trastuzumab and pertuzumab, The EC of the ADCC-enhancing anti-Her2 (anti-Her2) bispecific antibody 23C2 HER2-2 was higher than that of trastuzumab, T-DM1, and Expi HER2-1. 50 The EC of the combination of trastuzumab and pertuzumab was 0.01006 nM. 50 The EC of Expi HER2-1 was 0.06727nM. 50 The EC of T-DM1 was 0.08066 nM. 50 The EC value of trastuzumab was 0.08357 nM. 50 The cytolysis rate of the anti-Her2 bispecific antibody 23C2 HER2-2 was 0.07443 nM.

[0179] Example 11: Growth inhibition of BT474 Her2+++ tumor cells by anti-Her2 bispecific antibodies 23C2 Her2-2, trastuzumab, and pertuzumab were diluted to a final concentration of 3.2 μg / mL in DMEM / F12 medium (GIBCO, Catalog No. 11330-032) containing 2% FBS (fetal bovine serum, GIBCO, Catalog No. 10099-141) and then diluted 1:1 to nine concentrations: 1.6 μg / mL, 0.8 μg / mL, 0.4 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, 0.0125 μg / mL, and 0.00625 μg / mL. BT474 Her2+++ cells in logarithmic growth phase were incubated at a density of 1 × 10 5 The plated cells / mL were adjusted to 100 μL per well, and a blank well without cells was added as a control. The gradient diluted antibody was added at 50 μL per well. The plates were cultured at 37°C in a 5% CO2 incubator for 5 days. The culture medium was discarded, and 100 μL of CCK-8 (Dojindo Chemical, catalog number: CK04) was added as a working solution per well. The plates were incubated for 4-5 hours to allow color development, and then placed on a microplate reader (Thermo, model number: VarioskanFlash). The absorbance values ​​at a wavelength of 450 nm and a reference wavelength of 630 nm were read and recorded. The tumor cell growth inhibition rate was calculated.

[0180] As shown in Figure 6, the ADCC-enhancing anti-Her2 bispecific antibody 23C2 Her2-2 inhibited the growth of BT474 tumor cells by 78.38%, exceeding the 54.12% growth inhibition rate achieved by trastuzumab and the 53.7% growth inhibition rate achieved by the combination of trastuzumab and pertuzumab.

[0181] Example 12: Inhibitory effect of anti-Her2 bispecific antibodies on NCI-N87 Her2++ gastric cancer xenograft tumors in nude mice The in vivo efficacy of anti-Her2 bispecific antibodies was evaluated in a mouse xenograft model using NCI-N87 Her2++ gastric cancer cells (Cell Bank of the Committee for the Collection of Typical Cultures of the Chinese Academy of Sciences). NCI-N87 Her2++ gastric cancer cells were prepared at a concentration of 5 × 10 7 The volume was 0.1 mL / mouse at 100 cells / mL. Under sterile conditions, nude mice (provided by Changzhou Kavnese Laboratory Animal Co., Ltd., nude mice, 14-17 g, male, breeding environment: SPF) were inoculated into the right armpit. The diameter of the tumors transplanted into the nude mice was measured with a vernier caliper, and the tumors were 100-250 mm. 3 Once the animals reached maturity, they were divided into five groups. Group 1: Control Group 2: Expi Her2-1, 10mg / kg Group 3: 23C2 Her2-2, 5mg / kg Group 4: 23C2 Her2-2, 10mg / kg Group 5: Per+Tra (combination of trastuzumab and pertuzumab), 5+5 mg / kg.

[0182] Each treatment group received a predetermined dose by intravenous injection, administered twice a week for three consecutive weeks (six times). The administration volume of the two drugs in the combination group (Group 5) was 5 mL / kg of mouse body weight, while the administration volume for each of the other groups was 10 mL / kg of mouse body weight. Group 1 received 10 mL / kg of PBS (Hyclone, catalog number: sh30256.01) intravenously. In the administration group, trastuzumab was administered at least 30 minutes after pertuzumab administration.

[0183] The antitumor effect of the test substance was dynamically observed by measuring tumor diameter. Tumor volume was measured 2-3 times a week (see Table 8 for measurement times), and the mice were weighed and the data recorded. The general behavior of the mice was observed daily.

[0184] Detection indicators: The tumor volume (TV) was calculated using the formula TV = 1 / 2 × a × b 2 where a and b are the length and width, respectively.

[0185] In this experiment, administration began on day 0 and was administered a total of six times (days 0, 3, 7, 10, 14, and 17). By day 21 of the experiment, no animals had died. The average weight of mice in each group tended to increase (Figure 8). The drug had no obvious toxic effects.

[0186] The effects of Group 2 (10 mg / kg), Group 3 (5 mg / kg), Group 4 (10 mg / kg), and Group 5 (5 mg / kg + 5 mg / kg) on ​​the volume of NCI-N87 gastric cancer xenograft tumors in nude mice by day 21 are shown in Table 7 and Figure 7. 23C2 Her2-2 at a dose of 10 mg / kg was superior to Expi Her2-1 (10 mg / kg) and the Per+Tra combination group (5 mg / kg + 5 mg / kg) in inhibiting the growth of NCI-N87 gastric cancer xenograft tumors in nude mice. [Table 10]

Claims

1. The present invention relates to a method for producing an antibody against a HER2-expressing cell, the method comprising: (a) providing a first antigen-binding fragment that is monovalent and specifically binds to the ECD4 antigen of HER2 in a HER2-expressing cell; and (b) providing a second antigen-binding fragment that is monovalent and specifically binds to the ECD2 antigen of HER2 in a HER2-expressing cell, the first antigen-binding fragment being an scFv, and the first antigen-binding fragment comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 being identical to each other. the first antigen-binding fragment comprises the heavy chain CDR1 set forth in SEQ ID NO: 45, the heavy chain CDR2 set forth in SEQ ID NO: 46, the heavy chain CDR3 set forth in SEQ ID NO: 47, the light chain CDR1 set forth in SEQ ID NO: 48, the light chain CDR2 set forth in SEQ ID NO: 49, and the light chain CDR3 set forth in SEQ ID NO:

50.

2. 2. The bispecific antibody of claim 1 , wherein the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.

3. 2. The bispecific antibody of claim 1 , wherein the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 35 and the light chain variable region consists of the amino acid sequence of SEQ ID NO:

36.

4. The bispecific antibody of any one of claims 1 to 3, wherein the VH and VL of the first antigen-binding fragment are arranged in the order of VH-linker-VL from the N-terminus to the C-terminus.

5. The bispecific antibody of claim 1 , wherein the second antigen-binding fragment is a Fab.

6. 2. The bispecific antibody of claim 1, wherein the second antigen-binding fragment comprises a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO:

38.

7. 7. The bispecific antibody of claim 1, comprising an Fc operably linked to a first antigen-binding fragment and a second antigen-binding fragment.

8. The bispecific antibody of claim 7, wherein the Fc is derived from the Fc of human IgG1 or human IgG4.

9. The Fc has a modification, the modification comprising: (i) substitutions H435R and / or Y436F according to the EU numbering system; or / and (ii) The bispecific antibody according to claim 8, wherein the mutation site is knob-into-hole.

10. 8. The bispecific antibody of claim 7, wherein the Fc is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein a first antigen-binding fragment is operably linked to the first Fc polypeptide and a second antigen-binding fragment is operably linked to the second Fc polypeptide.

11. The bispecific antibody according to any one of claims 1 to 10, which is bivalent or multivalent.

12. 2. The bispecific antibody of claim 1 , selected from bivalent bispecific antibodies comprising a heavy chain comprising SEQ ID NO: 11, a heavy chain comprising SEQ ID NO: 13, and a light chain comprising SEQ ID NO:

15.

13. 13. The bispecific antibody of any one of claims 1 to 12, which has reduced aggregation compared to a bispecific antibody in which position 30 is not E and / or position 53 is not Y, and / or which does not have a clearly reduced binding affinity for the ECD4 antigen and / or the ECD2 antigen of HER2 compared to a bispecific antibody in which position 30 is not E and / or position 53 is not Y.

14. The bispecific antibody of any one of claims 1 to 12, which is defucosylated.

15. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 14 and a pharmaceutically acceptable excipient, diluent, encapsulating material, filler or buffer.

16. An isolated nucleic acid or a collection of isolated nucleic acids comprising at least one nucleic acid sequence encoding at least one antigen-binding fragment of the bispecific antibody of any one of claims 1 to 14.

17. 17. A vector or vector collection comprising one or more of the nucleic acids or nucleic acid collections of claim 16.

18. 18. An isolated cell comprising a nucleic acid or collection of nucleic acids according to claim 16, or a vector or collection of vectors according to claim 17.

19. 18. A method for producing a bispecific antibody according to any one of claims 1 to 14, comprising culturing a host cell under conditions suitable for expression of said bispecific antibody, said host cell comprising nucleic acid encoding said bispecific antibody, or the vector or vector collection of claim 17, and purifying said construct.

20. 15. Use of the bispecific antibody of any one of claims 1 to 14 or a pharmaceutical composition comprising said bispecific antibody in the manufacture of a medicament for treating a HER2-expressing tumor in a subject.

21. 21. The use of claim 20, wherein the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer, or skin cancer.

22. A pharmaceutical for treating a HER2-expressing tumor in a subject, comprising as an active ingredient the bispecific antibody according to any one of claims 1 to 14, or a pharmaceutical composition comprising said bispecific antibody.

23. 23. A pharmaceutical for treating a HER2-expressing tumor in a subject according to claim 22, wherein the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or skin cancer.

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