Antibody against human epidermal growth factor 3, method for producing the same, and use thereof

Abstract

This invention discloses an antibody against human epidermal growth factor 3 (HER3), a method for producing the same, and its use. The HER3 antibody specifically binds to HER3, is endocytotic by cells, and can be used to suppress diseases associated with HER3 expression (including overexpression) or affected by HER3 function, such as tumors.

Description

[Technical Field]

[0001] This application claims priority to the Chinese patent application filed on 13 June 2023, application number CN202310694043.3, the entire contents of which are incorporated herein by reference. This invention belongs to the biomedical field, and more specifically, it relates to an antibody against human epidermal growth factor 3, a method for producing the same, and the use of the same. [Background technology]

[0002] HER3 (ERRB3) belongs to the human epidermal growth factor receptor (EGFR) family, which also includes EGFR (ERRB1, HER1), HER2 (ERRB2), and HER4 (ERRB4). The extracellular domain of HER3 is classified into four domains, I to IV. Of these, I and III are leucine-rich B-helix regions responsible for ligand binding. II and IV are cysteine-rich regions, and of these, domain II also includes a dimeric arm essential for interaction with other receptors. The intracellular domain of HER3 includes the Juxta membrane region, kinase domain, and C-terminal tail.

[0003] HER3 ligands include neuregulin 1 (NRG-1) and neuregulin 2 (NRG-2). After the ligand binds to HER3, HER3 cannot form homodimers but instead forms heterodimers with other receptors, thereby inducing intracellular phosphorylation. Furthermore, HER3 has little intracellular tyrosine kinase activity and cannot initiate downstream signaling pathways on its own. Therefore, HER3 needs to bind to other receptor members of the EGFR family (e.g., EGFR and HER2) or non-EGFR family receptors (including MET factor receptor and FGFR2) to form heterodimers, participate in signal transduction, and initiate pathways related to cell proliferation or differentiation. HER3 facilitates heterodimerization with EGFR and HER2 but has low affinity for HER4. The heterodimer formed by HER3 and HER2 activates the phosphatidylinositol 3-kinase (PI3K) pathway. The activated PI3K / AKT signaling pathway is crucial for cancer cell survival and is a major cause of drug resistance in cancer cells.

[0004] HER3 is highly expressed in a variety of cancers, including breast, colon, lung, pancreatic, skin, gastric, ovarian, and melanoma, and its high expression is associated with disease progression and poor prognosis. Compared to other EGFR family members, HER3 alone does not exhibit oncogenicity when overexpressed. However, the heterodimer formed by HER3 and HER2 plays a crucial role in the development of many cancers. Studies have shown that upregulation of HER3 expression is one mechanism by which tumor cells evade EGFR family tyrosine kinase inhibition. Furthermore, signaling mediated by HER2 and HER3 heterodimers is associated with resistance to the EGFR tyrosine kinase inhibitor (TKI) gefitinib in head and neck cancers and breast cancers. In colorectal cancer, increased levels of the HER3 ligand NRG1 and HER2 and HER3 heterodimers induce resistance to the EGFR antibody cetoximab in patients. Therefore, HER3 is considered a major target in tumor therapy.

[0005] The development of monoclonal antibodies against HER3 is expected to be an effective treatment for recurrent tumors with high EGFR and HER2 expression. Because HER3 lacks clear kinase activity, anti-HER3 antibodies primarily act by blocking the interaction between HER3 and its ligand or between HER3 and other EGFR family receptors, or by recruiting immune cells to kill cancer cells. Currently, HER3-targeted antibodies are in clinical development and preclinical evaluation, but none have been launched. Furthermore, two-specific antibodies and antibody-drug conjugates (ADCs) targeting HER3 are also undergoing clinical trials. Daiichi Sankyo's antibody-drug conjugate, patritumab deruxtecan, is currently in Phase III clinical trials. Patritumab deruxtecan is a combination of the anti-human HER3 antibody patritumab and the small molecule drug DXd. Clinical trial results to date have confirmed its effectiveness in treating non-small cell lung cancer patients resistant to EGFR inhibitors. However, as HER3 becomes increasingly important in tumor progression and drug resistance, the development of HER3-targeted pharmaceutical compositions is of paramount importance. [Overview of the project]

[0006] The object of the present invention is to provide an antibody against human epidermal growth factor 3, a method for producing the same, and a method for using the same.

[0007] In a first aspect of the present invention, a HER3-specific antibody having a light chain variable region and a heavy chain variable region is provided. The antibody is The amino acid sequence of CDR1 in the light chain variable region was selected from the group consisting of SEQ ID NOs. 21, SEQ ID NOs. 30, SEQ ID NOs. 12, SEQ ID NOs. 18, and SEQ ID NOs. 27. The amino acid sequence of CDR2 in the light chain variable region was selected from the group consisting of SEQ ID NOs. 22, SEQ ID NOs. 31, SEQ ID NOs. 13, and SEQ ID NOs. 28. The amino acid sequences of CDR3 in the light chain variable region are SEQ ID NOs: 23, 32, 14, and 19. The amino acid sequence of CDR1 in the heavy chain variable region was selected from the group consisting of SEQ ID NOs: 24, 33, and 15. The amino acid sequence of CDR2 in the heavy chain variable region was selected from the group consisting of SEQ ID NOs. 25, SEQ ID NOs. 34, and SEQ ID NOs. 16. The amino acid sequence of CDR3 in the heavy chain variable region was selected from the group consisting of SEQ ID NOs: 26, 35, 17, 20, and 29. In one or more embodiments, the antibody is antibody (a) (which may include humanized (h)HE4, mouse (m)HE4, chimeric (c)HE4, etc.), the amino acid sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 21, 22, and 23, respectively, and the amino acid sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 24, 25, and 26, respectively.

[0008] In one or more embodiments, the antibody is antibody(c) (which may include humanized HE6 / mouse HE6 / chimeric HE6, etc.), the amino acid sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 30, 31, and 32, respectively, and the amino acid sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 33, 34, and 35, respectively.

[0009] In one or more embodiments, the antibody is antibody(d) (which may include humanized HE1 / mouse HE1 / chimeric HE1, etc.), the amino acid sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 12, 13, and 14, respectively, and the amino acid sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 15, 16, and 17, respectively.

[0010] In one or more embodiments, the antibody is antibody(e) (which may include humanized HE2 / mouse HE2 / chimeric HE2, etc.), the amino acid sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 18, SEQ ID NO: 13, and SEQ ID NO: 19, respectively, and the amino acid sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 20, respectively.

[0011] In one or more embodiments, the antibody is antibody(f) (which may include humanized HE5 / mouse HE5 / chimeric HE5, etc.), the amino acid sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 27, 28, and 19, respectively, and the amino acid sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 15, 16, and 29, respectively.

[0012] In one or more embodiments, the antibody comprises a monoclonal antibody, a single-chain antibody (scFv), a domain antibody, a Fab fragment, an Fd fragment, an Fv fragment, and an F(ab')2 fragment.

[0013] In one or more embodiments, the antibody is a humanized antibody, a chimeric antibody, or a mouse antibody.

[0014] In one or more embodiments, the chimeric antibody has a light chain variable region and a heavy chain variable region of a mouse antibody, but the heavy chain constant region of the antibody is replaced with the human antibody IgG1 heavy chain constant region, and the light chain constant region of the antibody is replaced with the human kappa light chain constant region.

[0015] In one or more embodiments, the humanized antibody retains the mouse CDR regions for the antibody variable regions, but based on the mouse variable region sequences, the FR region sequences of its heavy chain variable region and light chain variable region are replaced with the sequences having the closest homology to the FR regions of the human antibody germline genes, and then the antibody is formed. Preferably, the heavy chain constant region of the humanized antibody is the heavy chain constant region of human antibody IgG1, and the light chain constant region is the human kappa light chain constant region.

[0016] In one or more embodiments, the light chain FR regions of the humanized antibody have FR1 shown at positions 1 to 23 of SEQ ID NO: 36, FR2 shown at positions 35 to 49, FR3 shown at positions 57 to 88, and FR4 shown at positions 98 to 108.

[0017] In one or more embodiments, the heavy chain FR regions of the humanized antibody have FR1 shown at positions 1 to 27 of SEQ ID NO: 37, FR2 shown at positions 36 to 47, FR3 shown at positions 60 to 96, and FR4 shown at positions 112 to 122.

[0018] In one or more embodiments, the antibody is an antibody (humanized antibody) having a light chain variable region with an amino acid sequence shown in SEQ ID NO: 36 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 37.

[0019] In one or more embodiments, the antibody is an antibody (mHE4) having a light chain variable region with an amino acid sequence shown in SEQ ID NO: 5 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 6.

[0020] In one or more embodiments, the antibody is an antibody (mHE6) having a light chain variable region with an amino acid sequence shown in SEQ ID NO: 9 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 10.

[0021] In one or more embodiments, the antibody is an antibody (mHE7) having a light chain variable region with an amino acid sequence shown in SEQ ID NO: 9 and a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 11.

[0022] In one or more embodiments, the antibody is an antibody (mHE1) having an amino acid sequence of a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2.

[0023] In one or more embodiments, the antibody is an antibody (mHE2) having an amino acid sequence of a light chain variable region shown in SEQ ID NO: 3 and a heavy chain variable region shown in SEQ ID NO: 4.

[0024] In one or more embodiments, the antibody is an antibody (mHE5) having an amino acid sequence of a light chain variable region shown in SEQ ID NO: 7 and a heavy chain variable region shown in SEQ ID NO: 8.

[0025] In one or more embodiments, the antibody further includes an antibody having the same function as the antibody described in the examples of the present invention, wherein the heavy chain variable region amino acid sequence has 85% or more homology (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) with any of the above heavy chain variable region amino acid sequences, and the light chain variable region amino acid sequence has 85% or more homology (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) with any of the above light chain variable region amino acid sequences.

[0026] In another aspect of the present invention, a polynucleotide encoding the antibody is provided.

[0027] In another aspect of the present invention, a construct (e.g., an expression vector) comprising the polynucleotide is provided.

[0028] In another aspect of the present invention, an antibody expression system is provided which includes the construct or which incorporates the exogenous polynucleotide into the genome.

[0029] In one or more embodiments, the antibody expression system is a cell expression system.

[0030] In another aspect of the present invention, a method for producing a HER3-specific antibody according to any one of the above is provided, comprising expressing the antibody using the antibody expression system under conditions suitable for antibody expression to obtain the antibody.

[0031] In one or more embodiments, the manufacturing method further includes purifying and separating the antibody.

[0032] In another aspect of the present invention, the use of any of the antibodies described above is provided for use in the production of antitumor drugs that specifically target HER3-expressing tumor cells, the production of antibody-drug conjugates or immune conjugates, the production of bifunctional or multifunctional antibodies, the production of reagents for diagnosing HER3-expressing tumors, or the production of chimeric antigen receptor-modified immune cells.

[0033] In another aspect of the present invention, an immunocomplex is provided comprising an antibody as described above, and a functional molecule linked thereto (including covalent bonding, coupling, attachment, and adsorption).

[0034] In one or more embodiments, the functional molecule includes, but is not limited to, tumor suppressor molecules, molecules targeting tumor surface markers, cytotoxins, radioisotopes, biologically active proteins, molecules targeting surface markers of immune cells or detectable markers, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or combinations thereof.

[0035] In one or more embodiments, the tumor suppressor molecule is an antitumor toxin or an antitumor cytokine.

[0036] In one or more embodiments, the molecule targeting the tumor surface marker is an antibody or ligand that binds to the tumor surface marker.

[0037] In one or more embodiments, the surface marker molecule targeting the immune cells is an antibody or ligand that binds to the immune cell surface marker.

[0038] In one or more embodiments, the antitumor toxin includes, but is not limited to, a toxin that acts on microtubule proteins, a toxin or derivative thereof that acts on DNA, or a compound or derivative thereof that acts on intracellular metabolism, transcription, translation, or signal transduction.

[0039] In one or more embodiments, the antitumor cytokine includes, but is not limited to, IL-2, IL-12, IL-15, IFN-β, TNF-α, or variants thereof.

[0040] In one or more embodiments, the antibody that binds to the tumor surface marker is an antibody that recognizes an antigen other than HER3, the other antigen being EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin18.2, Claudin6, WT1, NY-ESO-1, MAGE3, CD47, ASGPR1, or CDH16.

[0041] In one or more embodiments, the antibody that binds to the immune cell surface marker comprises the antigen CD3, CD4, CD8, PD-1, PD-L1, LAG-3, TIM-3, 4-1BB, OX40, ICOS, B7-H3, CD16, CTLA-4, TIGIT, VISTA, GITR, CD27, SIRPα, CD32, CD64, ILT2, ILT4, NKG2A, NKG2D, or KIR.

[0042] In one or more embodiments, the toxin acting on the microtubule protein includes monomethyl auristatin (including its derivatives) and maytansinoid (including its derivatives).

[0043] In one or more embodiments, the toxin acting on the DNA includes DXd (including its derivatives), duocarmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), and SN-38 (including its derivatives).

[0044] In one or more embodiments, the toxin further comprises related compounds and derivatives thereof that act on other functions within tumor cells, such as metabolism, transcription, translation, and signal transduction.

[0045] In one or more embodiments, the antitumor toxin is linked to the antibody via a linker, the linker including, but not limited to, a maleimide-GGFG peptide linker, mc-Val-Cit-PAB (maleimidocaproyl valine-citrulline para-aminobenzyloxycarbonyl), or mc-Val-Ala-PAB (maleimidocaproyl valine-alanine para-aminobenzyloxycarbonyl).

[0046] In one or more embodiments, the detectable marker includes, but is not limited to, a fluorescent marker or a color-developing marker.

[0047] In one or more embodiments, the intracellular signaling region includes, but is not limited to, the CD3ζ chain, the FcεRIγ tyrosine activation motif, and the intracellular signaling regions of the co-stimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, and CD40.

[0048] In one or more embodiments, the transmembrane region includes, but is not limited to, a transmembrane region of CD8 or CD28.

[0049] In another aspect of the present invention, a pharmaceutical composition (including a diagnostic composition such as a diagnostic reagent) comprising the antibody or the immune complex described in any of the above is provided.

[0050] In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0051] In another aspect of the present invention, the use of the antibody, the immune complex, or the pharmaceutical composition in the manufacture of a formulation, reagent kit, or pharmaceutical kit for the diagnosis or treatment of a tumor is provided, wherein the tumor is a tumor that expresses HER3.

[0052] In one or more embodiments, the tumor includes, but is not limited to, breast cancer, colon cancer, lung cancer, pancreatic cancer, skin cancer, gastric cancer, ovarian cancer, and melanoma.

[0053] In another aspect of the present invention, a reagent kit or pharmaceutical kit comprising the antibody, the immune complex, or the pharmaceutical composition is provided.

[0054] Other aspects of the present invention will be apparent to those skilled in the art through the disclosure herein. [Brief explanation of the drawing]

[0055] [Figure 1] The results of detecting serum titers of the immunized mice according to the present invention using ELISA and FACS are shown. [Figure 2] The ELISA detection results of the chimeric antibody of the present invention against human and monkey HER3 are shown. [Figure 3] The FACS detection results of the chimeric antibody of the present invention against human and monkey HER3 are shown. [Figure 4] The results of antigen-binding epitope experiments between the chimeric antibody of the present invention and patritumab are shown. [Figure 5] The detection results for endocytosis of the chimeric antibody of the present invention are shown. [Figure 6]The present invention demonstrates the killing function of the chimeric antibody-drug conjugate against tumor cells. [Figure 7] The ELISA detection results for the humanized antibody of the present invention against human and monkey HER3 are shown. [Figure 8] The FACS detection results for the humanized antibody of the present invention are shown. [Figure 9] This invention demonstrates the humanization of the present invention and the circumstances under which patrizumab binds to different subdomains of HER3. [Figure 10] This demonstrates the binding ability of the humanized antibodies of the present invention to EGFR, HER2, and HER4. [Figure 11] The results of cell imaging detection of endocytosis using the humanized antibody of the present invention are shown. [Figure 12] The FACS detection results for endocytosis of the humanized antibody of the present invention are shown. [Figure 13] This demonstrates the tumor-killing function of the humanized antibody-drug conjugate of the present invention. [Figure 14] The present invention demonstrates the antitumor effect of the antibody-drug conjugate in SW620 tumor-bearing mice. [Figure 15] The antibody-drug conjugate of the present invention demonstrates antitumor effects in DiFi-bearing tumor mice. [Figure 16] This shows the process from hybridoma production to obtaining humanized antibodies. [Modes for carrying out the invention]

[0056] The inventors, through thorough research, disclose a HER3-specific antibody that specifically binds to HER3 and is endocytotically absorbed into cells. This antibody can be used to suppress diseases related to HER3 expression (including overexpression) or diseases affected by HER3 function, such as tumors.

[0057] term As used herein, the terms “antibody” or “immunoglobulin” are used herein as general terms encompassing full-length antibodies, single-chain antibodies, and all parts, domains, or fragments thereof (including, but not limited to, antigen-binding domains or fragments). Furthermore, as used herein, the terms “sequence” (for example, in terms such as “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence”) should be understood to encompass both the generally relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a particularly limited interpretation is required herein.

[0058] The term "monoclonal antibody" refers to an antibody molecule manufactured from a single molecule. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

[0059] The term "humanized antibody" refers to a molecule that essentially possesses an antigen-binding site derived from a non-human immunoglobulin, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of human immunoglobulin. The antigen-binding site may include a complete variable domain fused to a constant domain, or may include only a complementation-determining region (CDR) transplanted into an appropriate framework region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble human immunoglobulin. Some morphologies of humanized antibodies retain the entire CDR sequence. Other morphologies have one or more modified CDRs relative to the original antibody.

[0060] "Sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the sequences. "Sequence homology" indicates the percentage of identical amino acids or those representing conserved amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are well known to those skilled in the art. Amino acid sequence homology is usually measured using sequence analysis software. For example, homology can be determined using the BLAST program in the NCBI database.

[0061] The "effective dose" of a drug refers to the amount necessary to induce a physiological change in the cells or tissues to which it is administered.

[0062] The "therapeutic effective dose" of a drug, such as a pharmaceutical composition, refers to the amount effective in achieving the desired therapeutic or preventive effect in the required dosage and duration. A therapeutic effective dose of a drug, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0063] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0064] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient it contains is effective, and which does not contain other ingredients that are unacceptably toxic to the recipient of the composition.

[0065] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0066] The term “treatment / prevention” (and its grammatical variations) refers to an attempt to alter the natural course of a disease in an individual under treatment, and may be carried out as a preventive or clinical intervention during a clinicopathological course. Expected effects in treatment include, but are not limited to, prevention of disease onset or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or reduction of disease status, and elimination or improvement of prognosis. In some embodiments, the antibodies of the present invention are used to delay disease formation or slow the progression of disease symptoms.

[0067] The term "detectable marker" refers to a marker that can be linked to an antibody and used to confirm the presence and amount of a specific target under test. Such "detectable markers" are not limited to enzymes, fluorescent labels, radionuclides, quantum dots, or colloidal gold. More specific options may include horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0068] In the present invention, the tumor is a HER3 tumor, and preferably includes, but is not limited to, breast cancer, colon cancer, lung cancer, pancreatic cancer, skin cancer, gastric cancer, ovarian cancer, melanoma, or a combination thereof.

[0069] antibody The present invention provides an anti-HER3 antibody having a light chain CDR1-CDR3 and a heavy chain CDR1-CDR3 as listed in Table 6 or Table 3. In a preferred embodiment, the anti-HER3 antibody has a light chain variable region and a heavy chain variable region as listed in Table 6 or Table 2. The present invention also includes an antibody in which the amino acid sequence of the heavy chain variable region has 85% or more homology (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) to the sequences listed in Table 6 or Table 2, and the amino acid sequence of the light chain variable region has 85% or more homology (e.g., 88%, 90%, 93%, 95%, 97%, or 99% or more) to the sequences listed in Table 6 or Table 2, and which has the same function or similar activity as the antibody described in the examples of the present invention.

[0070] The antigen-binding properties of an antibody are typically determined by three complementarity-determining regions (CDRs), which are arranged in an orderly manner with FR regions, and the FR regions do not directly participate in the binding reaction. These CDRs form a cyclic structure, and the β-sheets formed by the FRs between them are structurally close to each other, constituting the antigen-binding site of the antibody. The CDR regions are sequences of immunologically interesting proteins, and the antibody of the present invention includes sequence modifications targeting the CDR regions and framework regions.

[0071] The anti-HER3 antibody provided by the present invention can efficiently and specifically bind to human HER3 or monkey HER3, efficiently block the action of HER3, and efficiently deliver other substances bound to it to target cells. When it binds to HER3 on the cell surface, it is rapidly endocytotically introduced into the cell.

[0072] The present invention further comprises an anti-HER3 antibody fusion protein comprising a first domain of the antibody described in the present invention and a second domain that extends the half-life in the body and / or has binding activity to effector cells. The fusion protein may be a binding molecule that can specifically bind to cells expressing HER3.

[0073] In the second domain, the fragment used to extend the in vivo hemilapse period may include serum albumin or a fragment thereof, polyethylene glycol, a domain that binds to serum albumin (e.g., an anti-serum albumin antibody), a polyethylene glycol-liposome complex, and the like. In the second domain, the fragment having binding activity to effector cells may include an immunoglobulin Fc region, and is preferably selected from a human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations to alter Fc-mediated effector function, including one or more combinations selected from CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be one or more combinations selected from IgG, IgA1, IgA2, IgD, IgE, IgM, and the IgG may specifically be one or more combinations selected from IgG1, IgG2, IgG3, or IgG4 subtypes. The immunoglobulin Fc region contained in the antibody fusion protein enables the fusion protein to form a dimer, simultaneously extending the in vivo half-life of the fusion protein and increasing Fc-mediated related activity. In one specific embodiment of the present invention, the immunoglobulin Fc region may be the Fc region of human IgG1, more specifically a wild-type IgG1 Fc sequence, the sequence may be introduced with mutations that alter Fc-mediated effector function, for example, a) a mutation that alters Fc-mediated CDC activity, b) a mutation that alters Fc-mediated ADCC activity, or c) a mutation that alters Fc-mediated ADCP activity. Such mutations are described in the following literature: Leonard G Presta, Current Opinion in Immunology 2008, 20:460-470; Esohe E. Idusogie et al., J Immunol 2000, 164:4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hinton et al., J Immunol, 2006, 176:346-356.

[0074] In the anti-HER3 antibody fusion protein provided by the present invention, a linker peptide can be provided between the first domain and the second domain. The linker peptide may be a flexible polypeptide chain consisting of alanine (A) and / or serine (S) and / or glycine (G), and the length of the linker peptide may be 3 to 30 amino acids, preferably 3 to 9, 9 to 12, 12 to 16, or 16 to 20 amino acids, and in another specific embodiment of the present invention, the length of the linker peptide may be 8 or 15.

[0075] The present invention further provides isolated polynucleotides encoding the antibody or fusion protein of the present invention, the polynucleotides may be RNA, DNA, or cDNA, etc. Methods for providing the isolated polynucleotides should be known to those skilled in the art, and they may be obtained by production, for example, automated DNA synthesis and / or recombinant DNA technology, or isolated from suitable natural sources.

[0076] Constructs and antibody expression systems The present invention also provides constructs comprising isolated polynucleotides as described in the present invention. Methods for constructing the constructs should be known to those skilled in the art, and for example, the constructs may be obtained by methods such as in vitro recombinant DNA technology, DNA synthesis technology, or in vivo recombinant technology, and more specifically, by inserting the isolated polynucleotides into the multi-clonal sites of an expression vector. The expression vector in the present invention usually refers to various commercially available expression vectors well known in the art, and may be, for example, bacterial plasmids, phages, yeast plasmids, plant cell viruses, adenoviruses, mammalian cell viruses such as retroviruses, or other vectors. The vector may also include one or more regulatory sequences operably ligated to the polynucleotide sequence, and the regulatory sequences may include appropriate promoter sequences. The promoter sequence is usually operably ligated to a sequence encoding the amino acid sequence to be expressed. The promoter is any nucleotide sequence that exhibits transcriptional activity in a selected host cell, and includes mutation promoters, cleavage promoters, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence may further include a suitable transcription termination sequence that the host cell recognizes to terminate transcription. The terminator sequence is ligated to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that functions in a selected host cell can be used in the present invention.

[0077] Typically, a suitable vector may include an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers. For example, these promoters include, but are not limited to, eukaryotic promoters such as the E. coli lac or trp promoter, the λ phage PL promoter, the CMV pre-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, the Pihia yeast methanol oxidase promoter, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes can be used to provide phenotypic traits for selecting transformed host cells, and include, but are not limited to, dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) in eukaryotic cell cultures, or tetracycline or ampicillin resistance in E. coli. When the aforementioned polynucleotide is expressed, the expression vector may further contain an enhancer sequence. When an enhancer sequence is inserted into the vector, transcription is enhanced. The enhancer is a cis-acting element of DNA, usually consisting of about 10 to 300 base pairs, and acts on the promoter to enhance gene transcription.

[0078] The present invention further provides an antibody expression system comprising a construct described in the present invention or having an exogenous polynucleotide described in the present invention incorporated into the genome. Any cell suitable for expression by an expression vector can be used as a host cell, for example, the host cell may be a prokaryotic cell (such as a bacterial cell), a lower eukaryotic cell (such as a yeast cell), or a higher eukaryotic cell (such as a mammalian cell), and specifically includes, but is not limited to, one or more combinations selected from bacterial cells of Escherichia coli, Streptomyces; Salmonella; fungal cells such as yeast, filamentous fungi, and plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS, HEK293 cells, or Bowes melanoma cells. Methods for constructing the expression system should be known to those skilled in the art and may include, but is not limited to, one or more combinations of, microinjection, gene gun, electroporation, virus-mediated transformation, electron shock, calcium phosphate precipitation, etc.

[0079] immune complex The present invention also provides an immune complex comprising an antibody or a fusion protein as described in the present invention. The immune complex typically also includes a functional molecule linked to the antibody or fusion protein (including, but not limited to, covalently, coupling, attachment, or adsorption), the functional molecule including, but not limited to, a detectable marker, a cytotoxin, a radioisotope, a bioactive protein, a molecule targeting a tumor surface marker, a molecule suppressing a tumor, a molecule targeting a surface marker of an immune cell, or a combination thereof.

[0080] The method for producing the immune complex should be well known to those skilled in the art, and for example, the antibody and / or fusion protein may be linked to a functional molecule directly or via a spacer of appropriate length, and the linking method may be chemical crosslinking or genetic engineering fusion expression, thereby obtaining the immune complex.

[0081] For therapeutic purposes, therapeutic effector groups such as radioactive groups may be suitable. These radioactive groups are composed of or include cytotoxic groups such as radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 33 P, 35 S, 90 Y, 99 Tc, 111 ln, 123 l, 125 l, 131 l, 201 TI, 213 Bi), toxins or cytostatic agents.

[0082] The immune complex can include the antibody or fusion protein of the present invention and a detectable marker. The detectable marker includes, but is not limited to, fluorescent markers, chromogenic markers, protein tags, for example, enzymes, scavenger molecule families, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. Also, it may include one or more markers. The markers for labeling antibodies for detection and / or analysis and / or diagnosis purposes depend on the specific detection / analysis / diagnosis techniques and / or methods used, such as immunohistochemical staining (tissue) samples, flow cytometry, etc. Labels suitable for detection / analysis / diagnosis techniques and / or methods well-known in the art are well-known to those skilled in the art.

[0083] The antibody or fusion protein of the present invention can be coupled to a labeling group (labeled polypeptide) and then used, for example, for diagnostic purposes. Suitable labeling groups can be selected from radioisotopes (e.g., those described above) or groups containing radioisotopes, radionuclides, fluorescent groups (e.g., green fluorescent proteins such as GFP and RFP, dyes, rhodamine, fluorescein and its derivatives (e.g., FITC), anthocyanin dyes), enzyme groups (e.g., horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase), chemiluminescent groups, biotin groups, metal particles (e.g., gold particles), magnetic particles (e.g., those having a core containing magnetite (Fe3O4) and / or maghemite (Fe2O3)), and the polypeptide groups of the present invention.

[0084] The immune complex may include the antibody or fusion protein of the present invention and a molecule that targets a surface marker of an immune cell. The surface marker molecule of the targeted immune cell recognizes the immune cell and guides the antibody of the present invention to the immune cell. At the same time, the antibody of the present invention can target the immune cell to tumor cells, thereby utilizing the killing effect of the antibody of the present invention and inducing specific tumor killing by the immune cell.

[0085] The immune complex may include the antibody or fusion protein of the present invention, and a molecule that targets at least one tumor surface marker or a molecule that suppresses tumors. The molecule that suppresses tumors may be an antitumor cytokine or an antitumor toxin. For example, the cytokine may be, but is not limited to, IL-2, IL-12, IL-15, IFN-β, TNF-α, etc. The molecule that targets the tumor surface marker may, for example, exert a synergistic effect with the antibody of the present invention to more accurately target tumor cells.

[0086] The immune complex may be a chimeric antigen receptor (CAR) that can be expressed on immune cells. The terms "immune cells" and "immune effector cells" are interchangeable and include T lymphocytes, NK cells, or NKT cells, preferably NK cells and T lymphocytes. The chimeric antigen receptor generally comprises a sequentially linked extracellular binding region, a transmembrane region, and an intracellular signaling region, where the extracellular binding region contains the antibody or fusion protein of the present invention. The design of the transmembrane and intracellular signaling regions based on CAR technology is well known in the art: for example, the transmembrane region may employ a transmembrane region of a molecule such as CD8 or CD28, and the intracellular signaling region may employ an immune receptor tyrosine activation motif (ITAM) CD3ζ chain or an intracellular signaling region of FcεRIγ tyrosine activation continuation and co-stimulatory signaling molecule such as CD28, CD27, CD137, CD134, MyD88, or CD40. More specifically, first-generation CAR T lymphocytes are defined as T lymphocytes whose intracellular signaling region contains only ITAM, and in this case, the parts of the chimeric antigen receptor are linked in the following format: scFv-TM-ITAM, which can induce an antitumor cytotoxic effect. Second-generation CAR T lymphocytes have an intracellular signaling region of CD28 or CD137 (also known as 4-1BB) added, and in this case, the parts of the chimeric antigen receptor are linked in the following format: scFv-TM-CD28-ITAM or scFv-TM-CD137-ITAM. The B7 / CD28 or 4-1BBL / CD137 co-stimulatory effect generated in the intracellular signaling region causes sustained proliferation of T lymphocytes, which in turn increase the secretion levels of cytokines such as IL-2 and IFN-γ, and enhance the survival time and antitumor effect of CAR T cells in the body. Third-generation CAR T lymphocytes In T lymphocytes, each portion of the chimeric antigen receptor is linked in the form of scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improving the survival time of CAR T cells in the body and their antitumor effect.

[0087] According to the above, a chimeric antigen receptor produced using the antibody or fusion protein of the present invention may include an extracellular binding region, a transmembrane region, and an intracellular signaling region sequentially linked as follows: the antibody or fusion protein of the present invention, CD8 and CD3ζ; the antibody or fusion protein of the present invention, CD8, CD137 and CD3ζ; the antibody or fusion protein of the present invention, the transmembrane region of the CD28 molecule (CD28a), the intracellular signaling region of the CD28 molecule (CD28b) and CD3ζ; or the antibody or fusion protein of the present invention, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137 and CD3ζ.

[0088] By expressing the aforementioned chimeric antigen receptor on the surface of immune effector cells, it becomes possible to utilize the killing effect of the antibody of the present invention while simultaneously enabling the immune effector cells to exert a highly specific cytotoxic effect against tumor cells that express HER3.

[0089] In a preferred embodiment of the present invention, the antibody of the present invention can be linked to a tumor suppressor molecule, preferably an antitumor toxin, which includes toxins acting on DNA such as DXd, duocalmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), related compounds such as SN-38 and their derivatives; toxins acting on tubulin such as monomethyl auristatin related compounds and their derivatives, maytansinoid related compounds and their derivatives; and related compounds and their derivatives that act on other intracellular functions such as metabolism, transcription, translation, and signal transduction. The present invention also includes analogues, isomers, and precursors of these small molecule compounds as toxins.

[0090] Pharmaceutical compositions and reagent kits The present invention also provides a pharmaceutical composition comprising the anti-HER3 antibody of the present invention, a fusion protein of the anti-HER3 antibody of the present invention, or the immune complex of the present invention.

[0091] The aforementioned pharmaceutical composition may further include various carriers pharmaceutically acceptable in the art. These pharmaceutically acceptable carriers are nontoxic to the recipient at the dose and concentration used, and specifically include buffers such as acetates, tris, phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, benzethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, p-hydroxybenzoic acid hydrocarbons (such as methylparaben or propylparaben), 1,2-dihydroxybenzene, resorcinol, cyclohexanol, 3-pentanol, and cresol, etc.); and serum albumin, gelatin, or immunoglobulins. This includes, but is not limited to, proteins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, tonicity modifiers such as trehalose and sodium chloride, sugars such as sucrose, mannitol, trehalose, or sorbitol, surfactants such as polysorbate, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEE®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutical preparations used for internal administration are generally sterile, and methods for achieving sterility of pharmaceutical preparations should be well known to those skilled in the art, and can be achieved, for example, by sterile filter filtration. Those skilled in the art can select an appropriate pharmaceutically acceptable carrier according to the required dosage form of the pharmaceutical composition and manufacture it into different dosage forms. For example, the pharmaceutical composition of the present invention includes, but is not limited to, various dosage forms such as tablets, injections, and lyophilized agents.

[0092] In the pharmaceutical composition, the content of the fusion protein and immune complex is usually an effective amount, and the content of the active ingredient corresponding to this effective amount can be determined based on the target of treatment and a specific method of administration. For example, based on the total mass of the pharmaceutical composition, the content of the fusion protein and immune complex may be in the range of approximately 0.01-99%, 0.1-70%, 1-30%, 0.01-0.05%, 0.05-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-1%, 1-3%, 3-5%, 5-10%, 10-20%, 20-30%, 30-50%, 50-70%, or 70-99%.

[0093] The fusion protein, immune complex, and pharmaceutical composition of the present invention can be administered as a single active ingredient or in combination therapy, i.e., in combination with other drugs. For example, the combination therapy can be in combination with at least one other antitumor drug. Alternatively, the combination therapy may be in combination with an antibody that targets another tumor-specific antigen, where the antibody that targets the other tumor-specific antigen is one or more antibodies selected from anti-EGFR antibody, anti-VEGF antibody, anti-HER2 antibody, or anti-Claudin18A2 antibody, and the inhibitor may preferably be a monoclonal antibody.

[0094] The present invention also provides a detection kit comprising an antibody, fusion protein, or immune complex described in the present invention. The kit may optionally include a container, a control (negative or positive control), a buffer, an auxiliary agent, etc., which a person skilled in the art can select according to the specific circumstances. The kit may also include instructions for use by a person skilled in the art.

[0095] The present invention further provides a detection method for detecting the HER3 protein using the antibody, which includes, but is not limited to, qualitative detection, quantitative detection, and localization detection. Specifically, the detection method includes, but is not limited to, immunofluorescence, immunohistochemistry, and radioimmunoassay.

[0096] A method for detecting the presence or absence of HER3 protein in a sample includes the steps of contacting the sample with the antibody of the present invention and observing whether an antibody complex is formed, the presence or absence of HER3 protein in the sample if an antibody complex is formed. The sample may be a cell and / or tissue sample, and the sample may be immobilized or lysed in a medium, and the level of HER3 protein in the immobilized or lysed sample is detected. In some embodiments, the sample to be detected may be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is further linked to a fluorescent dye, chemical, polypeptide, enzyme, isotope, tag, etc., which can be used for detection or detected by other reagents.

[0097] use The present invention also provides the use of the antibodies, fusion proteins, immune complexes, or pharmaceutical compositions of the present invention in the manufacture of pharmaceuticals for use in the diagnosis, treatment, or prevention of diseases associated with cells expressing (or overexpressing) HER3.

[0098] The "therapeutic effective amount" of the fusion protein, immune complex, or pharmaceutical composition provided by the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of injury or disability due to disease pain. For example, in the treatment of HER3-related tumors (including ovarian cancer, endometrial cancer, breast cancer, cervical cancer, etc.), the "therapeutic effective amount" preferably inhibits cell proliferation or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80% compared to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system to predict efficacy against human tumors. Alternatively, it can be evaluated by testing the cell proliferation inhibitory ability, which can be measured in vitro by tests known to those skilled in the art. The therapeutic effective amount of the fusion protein, immune complex, or pharmaceutical composition can usually reduce tumor size or otherwise alleviate the symptoms of the subject. Those skilled in the art can select an appropriate therapeutic dose depending on the situation, which may be determined, for example, based on the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. The prescription of treatment (e.g., the determination of the dosage) may be determined by a physician, and factors usually considered include, but are not limited to, the disease being treated, the individual patient's circumstances, the site of delivery, the method of administration, and other factors. A preventive dose refers to the amount effective in achieving the desired preventive effect at the required dosage and time. Generally, since preventive doses are administered to the subject before the onset of the disease or in the early stages of the disease, a "preventive dose" is often less than a "therapeutic dose," but this is not always the case.

[0099] The beneficial technical effects of this invention include the following: Currently, no anti-HER3 monoclonal antibodies are listed in this field, and at the same time, current clinical trial results indicate that the efficacy of anti-HER3 monoclonal antibodies is not significant. The anti-HER3 antibody of the present invention has high affinity and exhibits ideal antigen-binding activity.

[0100] The anti-HER3 antibody of the present invention can be rapidly and efficiently endocytized into cells and manufactured as an ADC drug or a bispecific antibody, thereby exerting its effects in tumor therapy. Clearly, a bispecific antibody or antibody-drug conjugate (ADC) against HER3 is more promising.

[0101] The antibody of the present invention, for example, hHE4-G1W, binds to different epitopes of HER3 compared to the control antibody patritumab, providing a novel targeted antibody for clinical use.

[0102] Compared to the control antibody patritumab, hHE4-G1W exhibits higher affinity for HER3-expressing tumor cells, faster intracellular endocytosis, and high affinity target binding to HER3-expressing tumor cells even after small molecule coupling, resulting in optimal cytotoxicity.

[0103] The present invention will be described in more detail below with reference to specific examples. These examples are provided solely for illustrative purposes and not to limit the present invention, and it should be understood that the scope of the present invention is not limited by these examples. Experimental methods in the following examples that do not specify concrete conditions generally follow the usual conditions described in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual, 3rd Edition" (Science Press), or the conditions recommended by the manufacturer.

[0104] Example 1: Production of animals used for hybridoma fusion A fusion protein of the extracellular region of human HER3 with a His tag (hHER3-ECD-His) was used as an immunogen to immunize multiple SJL mice (mouse numbers: 10886, 10888, 10889, 10890, 10891, 10892, 10893, 10894, and 10895) by intraperitoneal administration to stimulate antibody production against human HER3. Serum titers were detected using ELISA. First, the hHER3-ECD-His protein was adsorbed onto a 96-well ELISA plate, then gradient-diluted serum was added, and the plate was incubated at room temperature for 1 hour before being washed. Next, HRP-labeled rabbit anti-mouse full-length secondary antibody (Thermo Fisher) was added, incubated at room temperature for 1 hour, and then the ELISA plate was washed. Finally, the plate was stained with TMB, and the absorbance at 450 nm was recorded using a microplate reader. As shown in Figures A and B of Figure 1, the mouse serum titer reached 1:1000K or higher at the protein level.

[0105] Next, serum titers were detected using FACS with A375 cells expressing HER3. A375 cells were digested with trypsin, collected by centrifugation, resuspended in PBS, counted, added to a 96-well plate, centrifuged, the supernatant removed, and then immunosuppressed mouse serum from each group was added. The cells were cultured at room temperature for 30 minutes, washed twice, and PE-labeled goat anti-mouse full-length secondary antibody (Jackson) was added. After culturing for 30 minutes under light shielding, the cells were washed once, resuspended in PBS, and detected using a flow cytometer. As shown in Figures C and D of Figure 1, at the cellular level, the titer of 10890 mouse serum reached a maximum of 182K, and the titers of all remaining mice reached 1:20K or higher, indicating that antibodies produced in mouse serum can specifically recognize HER3 expressed on the surface of A375 cells.

[0106] Based on the results of serum titer detection, it was confirmed that mice immunized with human HER3 extracellular domain protein produced anti-human HER3 antibodies, and based on the antibody titer results, mice 10889 and 10890 were selected for hybridoma fusion.

[0107] Example 2: Acquisition of high-affinity antibodies Using electrofusion, splenic lymphocytes from immune mice were fused with myeloma cells SP2 / 0 to obtain hybridoma cells that secreted specific anti-human HER3 antibodies and could proliferate indefinitely. Subcloning screening of the hybridomas yielded multiple anti-human HER3-specific antibodies. ELISA and FACS were performed on a large number of these antibodies using the same method as in Example 1, and five antibodies with high affinity were screened. Experimental results using human HER3 or cyno HER3 as antigens are shown in Table 1.

[0108] [Table 1]

[0109] The results showed that the five subclones specifically bound to human and monkey HER3 antigens and exhibited good binding efficacy.

[0110] Example 3, Antibody Sequence mRNA (Omega) was extracted from the five hybridoma monoclonal cells obtained in Example 2 and reverse transcribed into cDNA (TAKARA). The heavy and light chain variable regions of the antibody gene were amplified from the reverse transcribed cDNA using PCR, ligated to the pMDTM18-T vector (TAKARA), and then transformed into competent cells DH5α. The heavy and light chains of the monoclonal antibody were obtained by sequencing. Tables 2 and 3 show the amino acid sequences of the variable region and CDR of the anti-human HER3 mouse monoclonal antibody, respectively. Sequence analysis revealed that amino acids 73-75 "NKS" in the mHE6 heavy chain variable region are glycosylation sites. Therefore, in the subsequent construction of a chimeric antibody, the mutation of "N" at position 73 to "D" was named mHE7 heavy chain, and the mHE7 light chain variable region was made identical to that of the mHE6 light chain variable region.

[0111] [Table 2]

[0112] [Table 3]

[0113] Example 4: Expression of Chimeric Antibodies Using PrimeSTAR@Max DNA Polymerase (TAKARA), the heavy chain and light chain variable region nucleic acid genes of the antibody obtained in Example 3 were PCR-cloned and combined with the encoding nucleic acid genes of the human antibody IgG1 heavy chain constant region (GenBank accession number for its amino acid sequence: AXN93653.1) and the human kappa light chain constant region (GenBank accession number for its amino acid sequence: AWK57456.1), respectively, to form the full-length heavy chain and light chain genes. The target genes were isolated by agarose gel electrophoresis, and after obtaining the target genes using a gel recovery kit (Omega), they were ligated into expression vectors using the ClonExpress Ultra One Step Cloning Kit (Vazyme).

[0114] After verifying the correctness of the expression vector through sequence confirmation, chimeric antibodies were expressed, and chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE6-G1W, and cHE7-G1W were obtained for the heavy and light chains corresponding to mHE1-mHE6, respectively. The functions of the chimeric antibodies were then verified.

[0115] Example 5: Analysis of the binding ability of chimeric protein-binding antigens (ELISA) The binding affinity of chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE6-G1W, and cHE7-G1W to human and monkey HER3 extracellular region proteins was detected using ELISA. Human HER3-ECD-His protein and monkey HER3-ECD-His (Sino bio) were adsorbed onto ELISA plates, 5% skim milk powder was added, and the mixtures were incubated at room temperature for 1 hour. The ELISA plates were then washed, gradient-diluted antibodies were added, and the mixtures were incubated at room temperature for 1 hour. The ELISA plates were then washed again. Next, HRP-labeled goat anti-human Fc secondary antibody (Jackson) was added, and the mixtures were incubated at room temperature for 1 hour. Finally, the mixtures were colorimetrically treated with TMB, and the absorbance at 450 nm was recorded using a microplate reader.

[0116] As shown in Figure 2A, the affinity of all chimeric antibodies to the human HER3 protein was comparable to that of the control antibody patritumab (derived from patent WO2007077028). However, Figure 2B shows the EC binding of cHE6-G1W and cHE7-G1W to the monkey HER3 protein. 50 However, it has been shown to be 3 to 4 times lower compared to patrizumab.

[0117] Example 6: Analysis of binding ability of chimeric protein-binding antigens (FACS) The binding of chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE6-G1W, and cHE7-G1W to human HER3 and monkey HER3 cells was detected by FACS. Human HER3-overexpressing CHO cells and monkey HER3-overexpressing CHO cells were centrifuged, resuspended in PBS, counted, added to a 96-well plate, centrifuged again, the supernatant was removed, gradient-diluted antibodies were added, and the cells were cultured at 4°C for 30 minutes. The cells were then washed twice, PE-labeled goat anti-human IgG Fc-labeled secondary antibody (Jackson) was added, and the cells were cultured at 4°C under light-shielding conditions for 30 minutes. The cells were then washed once, resuspended in PBS, and detected using a flow cytometer.

[0118] As shown in Figure 3A, the chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W, and cHE5-G1W bind to human HER3. 50 Compared to patritumab, the affinity was confirmed to be 2 to 7 times higher, and cHE6-G1W and cHE7-G1W showed similar affinity to patritumab.

[0119] As shown in Figure 3B, cHE4-G1W is more effective against EC15 in monkey HER3 compared to patrizumab. 50 While the value doubled, other chimeric antibodies showed a significant decrease in affinity for monkey HER3.

[0120] Example 7: Affinity analysis of chimeric antibodies The affinity of six chimeric antibodies to human HER3-ECD-His and monkey HER3-ECD-His proteins was detected using Octet. The results are shown in Table 4.

[0121] Based on Ka values, all six chimeric antibodies showed higher binding rates to human HER3-ECD-His (hHER3-ECD-His) and monkey HER3-ECD-His (cyno HER3-ECD-His) proteins than patritumab.

[0122] Based on Kd values, cHE4-G1W showed slower dissociation rates with both hHER3-ECD-His protein and cyno HER3-ECD-His protein than patritumab. However, while cHE6-G1W and cHE7-G1W showed slower dissociation rates with hHER3-ECD-His protein than patritumab, their dissociation rates with cyno HER3-ECD-His protein were faster than patritumab.

[0123] Based on the KD values, the affinity of cHE4-G1W, cHE6-G1W, and cHE7-G1W to hHER3-ECD-His is superior to that of patrizumab. Furthermore, cHE6-G1W and cHE7-G1W have similar affinity to both the hHER3-ECD-His protein and the cyno-HER3-ECD-His protein, indicating that removing the glycosylation site of cHE6-G1W does not affect its affinity to hHER3-ECD-His and cyno-HER3-ECD-His.

[0124] [Table 4]

[0125] Example 8: Thermal stability analysis of chimeric antibodies Dynamic light scattering (DLS) was used to detect the thermal stability of six chimeric antibodies and patrizumab. The DLS (Wyatt) instrument detects light scattering when the antibodies are heated from 25°C to 85°C, and the thermal stability of the antibodies is determined by the polymerization temperature T. agg The value obtained was obtained.

[0126] The results are shown in Table 5, and the polymerization temperature T of the six chimeric antibodies was determined by detection. agg It was confirmed that all values ​​exceeded 60°C. Here, the T values ​​for cHE1-G1W, cHE2-G1W, cHE4-G1W and cHE5-G1W were determined. agg All of these values ​​were higher than those of the control antibody, patritumab, indicating that the thermal stability of these four chimeric antibodies is superior to that of patritumab.

[0127] [Table 5]

[0128] Example 9: Binding epitope analysis of chimeric antibodies Furthermore, the epitopes to which cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE7-G1W, and patrizumab bind to human HER3 were analyzed using Octet analysis. First, the AMC sensor was bound to 10 μg / mL mouse Fc-tagged human HER3-ECD protein. Next, the primary antibody was bound until the signal was saturated, and finally, the secondary antibody was bound, and it was observed whether the signal value increased or not. An increase in the signal value indicates that the primary and secondary antibodies are bound to different epitopes of human HER3, while no change in the signal value indicates that the primary and secondary antibodies are bound to the same epitope of human HER3.

[0129] The detection results using this method are shown in Figure 4. cHE1-G1W, cHE2-G1W, cHE4-G1W, and cHE5-G1W all bind to the same epitope, while cHE7-G1W binds to a different epitope. None of these antibodies compete for binding to patritumab or human HER3, indicating that these antibodies have different binding sites on patritumab and human HER3.

[0130] Example 10: Analysis of cellular endocytosis after antigen binding of chimeric antibodies The purpose of this invention is to test whether cHE4-G1W, cHE5-G1W, and cHE7-G1W bind to human HER3 and are then endocytotically absorbed into cells together with human HER3. 10 nM antibodies were mixed with A375 cells overexpressing human HER3, cultured at 4°C for 1 hour, and the unbound antibodies were washed off. The samples were left to stand at 4°C after 0 hours, then transferred to a 37°C incubator for culture. The cells were collected after 1 hour, 2 hours, and 3 hours, respectively. PE-labeled goat anti-human Fc secondary antibody (Jackson) was added to the samples, and the cells were cultured at 4°C for 30 minutes, after which the unbound secondary antibodies were washed off. Next, detection was performed using a flow cytometer.

[0131] As shown in Figure 5, the analysis results indicate that cHE4-G1W, cHE5-G1, and cHE7-G1W all underwent endocytosis after binding to HER3 on the surface of A375 cells, with cHE7-G1W showing an endocytosis rate nearly equivalent to that of the control antibody, patrizumab.

[0132] Example 11: Production of a conjugate drug based on a chimeric antibody and analysis of its tumor-killing effect. Three chimeric antibodies, cHE4-G1W, cHE5-G1W, and cHE7-G1W, were prepared as antibody-drug conjugates, and their killing effects against tumor cells A375, which overexpress human HER3, and tumor cells OVCAR3, which express HER3, were detected. The chimeric antibodies were first cultured with TCEP (sigma), the interchain disulfide bonds were cleaved, and then maleimide-GGFG-DXd (MedChemExpress) was added for coupling. Finally, the conjugates were purified using a desalting column to remove excess small molecule drugs. The resulting antibody-drug conjugates were named cHE4-G1W-DXd, cHE5-G1W-DXd, cHE7-G1W-DXd, and patrizumab-DXd, respectively, with IgG-DXd used as the negative control. Analysis by hydrophobic interaction chromatography revealed the following ratios for cHE4 antibody to DXd: 1:7, cHE5 antibody to DXd: 1:6.9, cHE7 antibody to DXd: 1:7.7, patritumab antibody to DXd: 1:8, and IgG to DXd: 1:7.5.

[0133] Tumor cell lines A375 and OVCAR3, which overexpress human HER3, were seeded at 5000 cells / well in 96-well cell plates. Antibody-drug conjugates cHE4-G1W-DXd, cHE5-G1W-DXd, cHE7-G1W-DXd, patrizumab-DXd, and negative control IgG-DXd were gradient diluted and added to 96-well cell plates containing human HER3-overexpressing A375 and OVCAR3 cells, respectively, and cultured at 37°C for 5 days. Cell viability was detected using CellTiter-Glo (Promega), thereby obtaining the cytotoxic effect of the antibody-drug conjugates.

[0134] As shown in Figures 6A and 6B, in A375 cells overexpressing human HER3, cHE4-G1W-DXd, cHE5-G1W-DXd, and cHE7-G1W-DXd all showed significant killing effects, with cHE4-G1W-DXd showing twice the killing effect of the control antibody patritumab-DXd. In OVCAR3 cells, compared to the negative control IgG-DXd, cHE4-G1W-DXd, cHE5-G1W-DXd, and cHE7-G1W-DXd all showed killing effects, and were close to the killing effect of patritumab-DXd.

[0135] Example 12: Production of humanized antibodies Based on the experimental results of Examples 5 to 11, the chimeric antibody cHE4-G1W was selected for humanization. Analysis of the variable regions of the light and heavy chain sequences of the cHE4-G1W antibody using the IMGT database revealed that the CDR1, CDR2, and CDR3 regions, which play a crucial role in antigen binding, are as shown in Table 3. In humanization, the CDR region was retained, and the mouse-derived FR region was replaced with one that has the closest homology to the FR region of the human antibody germline gene, ultimately yielding a humanized antibody. The variable regions of its light and heavy chains are shown in Table 6, respectively.

[0136] [Table 6]

[0137] The amino acid sequences of the heavy and light chain variable regions of the hHE4 antibody were converted to nucleotide sequences, and after codon optimization, gene synthesis was performed. These sequences were then ligated to the constant region genes of the human IgG1 heavy and light chains, respectively, and constructed into expression vectors. After confirming the correctness of the sequencing results, the plasmids were extracted and transfected into ExpiCHO cells for expression. Purification with protein A yielded the humanized antibody hHE4-G1W. The process for obtaining the humanized antibody is shown in Figure 16.

[0138] Example 13: Affinity analysis of humanized antibodies The affinity of the humanized antibody hHE4-G1W to human HER3 and monkey HER3 was detected compared to the chimeric antibody cHE4-G1W using the ELISA method. Human HER3-ECD-His protein and monkey HER3-ECD-His were adsorbed onto ELISA plates, 5% skim milk powder was added, and the mixture was incubated at room temperature for 1 hour to block. The ELISA plates were then washed, gradient-diluted antibodies were added, and the mixture was incubated at room temperature for 1 hour, after which the ELISA plates were washed again. Next, HRP-labeled goat anti-human Fc secondary antibody (Jackson) was added, and the mixture was incubated at room temperature for 1 hour, after which the ELISA plates were washed. Finally, the mixture was colorimetrically treated with TMB, and the absorbance at 450 nm was recorded using a microplate reader. As shown in Figures 7A and 7B, the humanized hHE4-G1W antibody had the same binding ability to human and monkey HER3 as the chimeric antibody cHE4-G1W, and its affinity was superior to that of the control antibody patrizumab in both cases.

[0139] Example 14: Analysis of the antigen-binding ability of humanized antibodies By FACS, the difference in affinity between the humanized antibody hHE4-G1W and the chimeric antibody cHE4-G1W for cells expressing human HER3 and monkey HER3 was detected. The method was the same as in Example 6.

[0140] As shown in Figure 8, in all CHO cells overexpressing human HER3 (Figure A), original A375 (Figure C), or CHO cells overexpressing monkey HER3 (Figure B), the humanized antibody hHE4-G1W showed no change in cellular binding ability to human and monkey HER3 compared to the chimeric antibody cHE4-G1W. Furthermore, hHE4-G1W showed improved EC binding to human HER3 compared to the control antibody patrizumab. 50 The values ​​were 4 to 8 times better.

[0141] Example 15: Affinity analysis of humanized antibodies The affinity of the humanized antibody hHE4-G1W to human HER3-ECD-His and monkey HER3-ECD-His proteins was detected using Octet. As shown in Table 7, the results, based on Ka, Kd, ​​and KD values, showed no significant difference in affinity between the humanized antibody hHE4-G1W and the chimeric antibody cHE4-G1W to humans and monkeys. Furthermore, the KD value of hHE4-G1W was 10 times stronger than that of patrizumab.

[0142] [Table 7]

[0143] Example 16: Epitope Conjugation Analysis of Humanized Antibodies The domain location to which the humanized antibody hHE4-G1W binds to HER3 was detected using ELISA. According to the literature (Acta Crystallogr F Struct Biol Commun. 2021 Jul 1;77(Pt 7):192-201), the extracellular domain of human HER3 is divided into four structural domains: domain I is at amino acids 56-183, domain II is at amino acids 184-308, domain III is at amino acids 309-500, and domain IV is at amino acids 501-643. Fusion proteins of domains I-III (56-500) and I-IV (56-643) with mFc tags were expressed and purified, respectively, and used for ELISA detection. Goat anti-human Fc (Jackson) was adsorbed onto an ELISA plate for 3 hours. After washing the plate, 5% skim milk powder was added and incubated at room temperature for 1 hour. After washing the ELISA plate again, 2 μg / mL of hHE4-G1W and patrizumab were added and incubated at room temperature for 1 hour. The ELISA plate was washed again, gradient diluted mFc-tagged HER3 I-III domains (56-500) or HER3 I-IV domains (56-643) fusion proteins were added and incubated for 1 hour. Next, HRP-labeled rabbit anti-mouse Fc secondary antibody (Thermo Fisher) was added and incubated at room temperature for 1 hour, after which the ELISA plate was washed. Finally, the mixture was colorimetrically treated with TMB, and the absorbance at 450 nm was recorded using a microplate reader.

[0144] As shown in Figure 9, the hHE4-G1W antibody can bind to the I-IV domains of HER3, but not to the I-III domains, indicating that the hHE4-G1W antibody binds to the IV domain of HER3. On the other hand, patritumab can bind not only to the I-IV domains of HER3, but also to the I-III domains, indicating that the binding site of the patritumab antibody is located on the I-III domains.

[0145] Therefore, the hHE4-G1W antibody bound to different epitopes of patritumab and human HER3.

[0146] Example 17: Analysis of binding specificity of humanized antibodies To demonstrate that hHE4-G1W specifically binds to human HER3, we used ELISA to detect whether hHE4-G1W nonspecifically binds to the human epidermal growth factor receptor (EGFR) family members EGFR, HER2, and HER4. Human EGFR-ECD-His protein, human HER2-ECD-His protein, and human HER4-ECD-His protein were adsorbed onto ELISA plates, 5% skim milk powder was added, and the mixtures were incubated at room temperature for 1 hour to block. After blocking, the ELISA plates were washed. Next, 25 nM antibody was added after a 3-fold gradient dilution to 0.011 nM, and the mixtures were incubated at room temperature for 1 hour. The ELISA plates were then washed again. Subsequently, HRP-labeled goat anti-human Fc secondary antibody (Jackson) was added, and the mixtures were incubated at room temperature for 1 hour. Finally, the ELISA plates were stained with TMB, and the absorbance at 450 nm was recorded using a microplate reader.

[0147] As shown in Figures A-C of Figure 10, the results indicate that neither hHE4-G1W nor patrizumab binds to EGFR, HER2, or HER4, while hHE4-G1W specifically binds only to human HER3.

[0148] Example 18: Thermal stability analysis of humanized antibodies The thermal stability of the humanized antibody hHE4-G1W was detected using the DLS method. The method was the same as in Example 8. As shown in Table 8, the thermal stability of humanized hHE4-G1W increased from 69.29°C to 75.54°C compared to the chimeric antibody cHE4-G1W, indicating that the thermal stability of the humanized antibody hHE4-G1W is significantly superior to that of patrizumab.

[0149] [Table 8]

[0150] Example 19: Analysis of cell endocytosis after antigen binding of humanized antibodies Using a cell imaging microplate analyzer, the presence or absence of endocytosis after binding of hHE4-G1W, hHE4-G1W-DXd, patritumab, and patritumab-DXd to HER3 on the surface of tumor cell A375 was monitored. 100 nM of hHE4-G1W, hHE4-G1W-DXd, patritumab, and patritumab-DXd were each cultured with A375 cells overexpressing the full length of HER3 at 4°C for 30 minutes. After washing away unbound antibodies, PE-labeled goat anti-human Fc secondary antibody (Jackson) was added to the sample, and the cells were cultured at 4°C for 30 minutes to wash away unbound secondary antibodies. Samples in which no endocytosis occurred after antibody binding to the A375 cell surface were designated as 0-hour samples, and fluorescence imaging was performed using a Biotek Cytation 1 Imaging Reader. Subsequently, the cells were cultured in a 37°C incubator for 3 hours, and fluorescence imaging was performed again.

[0151] As shown in Figure 11, at 0 hours, fluorescence was observed on the surface of A375 cells, indicating that hHE4-G1W, hHE4-G1W-DXd, patritumab, and patritumab-DXd all bound to HER3 on the cell surface. After 3 hours of culture, the fluorescence on the cell surface decreased, and conversely, fluorescence accumulated inside the cells, indicating that after 3 hours, hHE4-G1W, hHE4-G1W-DXd, patritumab, and patritumab-DXd were endocytotically absorbed from HER3 bound to the surface of A375 cells.

[0152] Example 20: Analysis of cell endocytosis after antigen binding of humanized antibodies Using FACS, the endocytosis rates of the humanized antibody hHE4-G1W, the chimeric antibody cHE4-G1W4, and the control antibody patrizumab after binding to A375 cells expressing HER3 on the cell surface were detected. The method was as described in Example 10.

[0153] As shown in Figure 12, the endocytosis rates were the same for hHE4-G1W and cHE4-G1W4 after binding to HER3 on the surface of A375 cells and then culturing at 37°C for 3 hours, and the endocytosis rate was faster compared to the control antibody patrizumab.

[0154] Example 21: Production of a conjugate drug based on a humanized antibody and analysis of its tumor-killing effect. The killing effect of the humanized antibody hHE4-G1W conjugate drug against HER3-expressing HCC1569, A375, and JIMT-1 tumor cells was detected. The antibody-small molecule coupling method was as described in Example 11, and the results were analyzed by hydrophobic interaction chromatography. The ratios of cHE4-G1W-DXd, hHE4-G1W-DXd, patrizumab-DXd, negative control IgG-DXd, and conjugate drug antibody to small molecule were 1:7, 1:8, 1:7.8, and 1:8, respectively.

[0155] As shown in Figures 13A-C, the results showed that hHE4-G1W-DXd, cHE4-G1W-DXd, and patritumab-DXd all demonstrated significant killing effects against HCC1569 cells expressing HER3 or A375 cells and JIMT-1 cells overexpressing human HER3. Furthermore, hHE4-G1W-DXd showed significantly superior killing effects compared to patritumab-DXd in JIMT-1 cells overexpressing human HER3.

[0156] Example 22: Analysis of tumor-suppressing effects of a conjugate drug based on a humanized antibody against the tumor-bearing animal SW620. The antitumor effects of hHE4-G1W-DXd and cHE5-G1W-DXddXd in SW620 tumor-bearing mice were analyzed. SCID female mice were selected, and a tumor-bearing mouse model was constructed by subcutaneous injection of SW620 cells into the back. After model construction, the mice were randomly divided into nine groups, and a single dose was administered. Three groups served as negative controls. Of these, one group received saline (PBS), the other two received low-dose 3 mg / kg IgG-DXd and high-dose 6 mg / kg IgG-DXd, respectively. The experimental groups received low-dose 3 mg / kg and 6 mg / kg hHE4-G1W-DXd, cHE5-G1W-DXd, and patrizumab-DXd, respectively. The average number of antibody-bound small molecules DXd was 8. Body weight and tumor volume were measured in each group of mice.

[0157] The results in Figure 14 show that the experimental groups had an inhibitory effect on SW620 tumor growth compared to the PBS and IgG-DX control groups. However, with 3 mg / kg patritumab-DXd, clear tumor growth was observed 14 days after administration, while with 3 mg / kg hHE4-G1W-DXd, no tumor growth was observed at all after administration, and with 6 mg / kg hHE4-G1W-DXd, the tumor completely disappeared in one mouse after administration. This explains why hHE4-G1W-DXd shows a superior inhibitory effect on SW620 tumors compared to patritumab-DXd.

[0158] Example 23: Analysis of tumor-suppressive effects of humanized antibody-based conjugate drugs on tumor-bearing animals (DiFi). The antitumor effects of hHE4-G1W-DXd and cHE5-G1W-DXd in DiFi tumor-bearing mice were analyzed. SCID female mice were selected, and a tumor-bearing mouse model was constructed by subcutaneous injection of DiFi cells into the back. After model construction, the mice were randomly divided into nine groups and administered once a week for a total of three times. Three groups served as negative controls. Of these, one group received saline (PBS), the other two received low-dose 3 mg / kg IgG-DXd and high-dose 6 mg / kg IgG-DXd, respectively. The experimental groups received low-dose 3 mg / kg and high-dose 6 mg / kg hHE4-G1W-DXd, cHE5-G1W-DXd, and patrizumab-DXd, respectively. The average number of antibody-bound small molecules DXd was eight. Body weight and tumor volume were measured for each group of mice.

[0159] The results in Figure 15 explain that the experimental group had an inhibitory effect on DiFi tumor growth compared to the PBS and IgG-DX control groups. However, compared to 3 mg / kg hHE4-G1W-DXd and cHE5-G1W-DXd, the tumor suppression effect of 3 mg / kg patritumab-DXd was relatively weak, while the 6 mg / kg dose group approached the tumor suppression effect. This explains that hHE4-G1W-DXd and cHE5-G1W-DXd have an equivalent or better antitumor effect compared to patritumab-DXd against DiFi tumors.

[0160] The embodiments described above illustrate several embodiments of the present invention, and while their descriptions are specific and detailed, they should not be interpreted as limiting the scope of the patent for the present invention. Those skilled in the art can make some modifications and improvements without departing from the concept of the present invention, and all of these fall within the scope of protection of the present invention. Accordingly, the scope of protection of the patent for the present invention is defined by the appended claims. Furthermore, all documents referenced in the present invention are cited in this application as references, just as each document is cited individually as a reference.

Claims

1. A HER3-specific antibody having a light chain variable region and a heavy chain variable region, selected from the following. The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs: 21, 22, and 23, respectively, and the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs: 24, 25, and 26, respectively. Antibody (a), The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 30, 31, and 32, respectively, and the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs. 33, 34, and 35, respectively. (c) The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs: 12, 13, and 14, respectively, and the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs: 15, 16, and 17, respectively. Antibody (d), The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 18, SEQ ID NO: 13, and SEQ ID NO: 19, respectively, and the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 20, respectively. Antibody (e), The amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs: 27, 28, and 19, respectively, and the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NOs: 15, 16, and 29, respectively, in the antibody (f).

2. The aforementioned antibodies include monoclonal antibodies, single-chain antibodies, domain antibodies, Fab fragments, Fd fragments, Fv fragments, and F(ab') ２ Contains fragments, or The antibody according to claim 1, characterized in that the antibody is a humanized antibody, a chimeric antibody, or a mouse antibody.

3. The antibody according to claim 1, characterized in that the antibody is selected from the following. Antibodies relating to the amino acid sequence shown in SEQ ID NO: 36, or a light chain variable region having 85% or more homology to said sequence; or antibodies relating to the amino acid sequence shown in SEQ ID NO: 37, or a heavy chain variable region having 85% or more homology to said sequence; Antibodies relating to the amino acid sequence shown in SEQ ID NO: 5, or a light chain variable region having 85% or more homology to said sequence; or antibodies relating to the amino acid sequence shown in SEQ ID NO: 6, or a heavy chain variable region having 85% or more homology to said sequence; Antibodies relating to the amino acid sequence shown in SEQ ID NO: 9, or a light chain variable region having 85% or more homology to said sequence; or antibodies relating to the amino acid sequence shown in SEQ ID NO: 10, or a heavy chain variable region having 85% or more homology to said sequence; Antibodies relating to the amino acid sequence shown in SEQ ID NO: 9, or a light chain variable region having 85% or more homology to said sequence; or antibodies relating to the amino acid sequence shown in SEQ ID NO: 11, or a heavy chain variable region having 85% or more homology to said sequence; Antibodies having the amino acid sequence shown in SEQ ID NO: 1, or a light chain variable region having 85% or more homology to said sequence; or the amino acid sequence shown in SEQ ID NO: 2, or a heavy chain variable region having 85% or more homology to said sequence; Antibodies relating to the amino acid sequence shown in SEQ ID NO: 3, or a light chain variable region having 85% or more homology to said sequence; or antibodies relating to the amino acid sequence shown in SEQ ID NO: 4, or a heavy chain variable region having 85% or more homology to said sequence; An antibody comprising the amino acid sequence shown in SEQ ID NO: 7, or a light chain variable region having 85% or more homology to said sequence; or an antibody comprising the amino acid sequence shown in SEQ ID NO: 8, or a heavy chain variable region having 85% or more homology to said sequence.

4. A polynucleotide encoding an antibody according to any one of claims 1 to 3.

5. A construct comprising the polynucleotide described in claim 4.

6. An antibody expression system comprising the construct described in claim 5, or having an exogenous polynucleotide described in claim 4 incorporated into the genome.

7. A method for producing a HER3-specific antibody according to any one of claims 1 to 3, comprising expressing the antibody using the antibody expression system described in claim 6 under conditions suitable for the expression of the antibody, thereby obtaining the antibody.

8. Use of the antibody according to any one of claims 1 to 3, Manufacturing of antitumor drugs that specifically target tumor cells expressing HER3. Manufacturing of antibody-drug conjugates or immune conjugates, Manufacturing of bifunctional or multifunctional antibodies, Manufacturing of reagents for diagnosing tumors that express HER3, or Used in the production of chimeric antigen receptor-modified immune cells.

9. An immune complex comprising an antibody according to any one of claims 1 to 3, and a functional molecule linked thereto.

10. The immune complex according to claim 9, characterized in that the functional molecule includes a tumor suppressor molecule, a molecule that targets a tumor surface marker, a cytotoxin, a radioisotope, a biologically active protein, a molecule that targets a surface marker of an immune cell or a detectable marker, an extracellular hinge region, a transmembrane region and an intracellular signaling region based on chimeric antigen receptor technology, or a combination thereof.

11. The tumor suppressor molecule is either an antitumor toxin or an antitumor cytokine, or The molecule targeting the tumor surface marker is either an antibody or ligand that binds to the tumor surface marker, or The immune complex according to claim 10, characterized in that the surface marker molecule targeting the immune cells is an antibody or ligand that binds to the immune cell surface marker.

12. The aforementioned antitumor toxin includes a toxin that acts on microtubule proteins, a toxin that acts on DNA or a derivative thereof, a compound that acts on intracellular metabolism, transcription, translation or signal transduction or a derivative thereof, or The antitumor cytokines include IL-2, IL-12, IL-15, IFN-β, TNF-α, or their variants, or The antibody that binds to the tumor surface marker is an antibody that recognizes an antigen other than HER3, and the other antigen includes EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin18.2, Claudin6, WT1, NY-ESO-1, MAGE3, CD47, ASGPR1, or CDH16, or The immune complex according to claim 11, characterized in that the antigen to which the antibody binding to the immune cell surface marker binds includes CD3, CD4, CD8, PD-1, PD-L1, LAG-3, TIM-3, 4-1BB, OX40, ICOS, B7-H3, CD16, CTLA-4, TIGIT, VISTA, GITR, CD27, SIRPα, CD32, CD64, ILT2, ILT4, NKG2A, NKG2D, or KIR.

13. The toxins that act on the microtubule proteins include monomethyl auristatin, maytansinoids, or The immune complex according to claim 12, characterized in that the toxin acting on the DNA comprises DXd, duocalmycin, calicheamicin, pyrrolobenzodiazepine, and SN-38.

14. The immunocomplex according to claim 11 or 12, wherein the antitumor toxin is linked to the antibody according to any one of claims 1 to 3 via a linker, and the linker comprises a maleimide-GGFG linker, mc-Val-Cit-PAB, or mc-Val-Ala-PAB.

15. A pharmaceutical composition comprising the antibody described in any one of claims 1 to 3 or the immune complex described in any one of claims 9 to 14.

16. Use of an antibody according to any one of claims 1 to 3, an immune complex according to any one of claims 9 to 14, or a pharmaceutical composition according to claim 15 in the manufacture of a formulation, reagent kit, or pharmaceutical kit for the diagnosis or treatment of a tumor, wherein the tumor is a tumor that expresses HER3.

17. The use according to claim 16, characterized in that the tumor includes breast cancer, colon cancer, lung cancer, pancreatic cancer, skin cancer, gastric cancer, or ovarian cancer.

18. The use according to claim 17, characterized in that the skin cancer is melanoma.

19. A reagent kit or pharmaceutical kit comprising an antibody according to any one of claims 1 to 3, an immune complex according to any one of claims 9 to 14, or a pharmaceutical composition according to claim 15.

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