Human epidermal growth factor 3 antibody, preparation method therefor, and use thereof

Abstract

A human epidermal growth factor 3 (HER3) antibody, a preparation method therefor, and a use thereof. The HER3 antibody can specifically bind to HER3 and be endocytosed by cells, and can be used for inhibiting diseases related to HER3 expression (including overexpression) or affected by the function of HER3, such as tumors.

Description

Antibody to human epidermal growth factor 3, preparation method and application thereof

[0001] This application claims priority to patent application No. CN 202310694043.3, filed on June 13, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of biomedicine, and more specifically, relates to antibodies against human epidermal growth factor 3, a preparation method thereof, and applications thereof. Background Art

[0003] 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 divided into four domains, I to IV. Domains I and III are leucine-rich B-helical regions responsible for ligand binding. Domains II and IV are cysteine-rich regions, with domain II also containing a dimerization arm necessary for interaction with other receptors. The intracellular domain of HER3 includes the Juxta membrane region, the kinase structure, and the C-terminal tail.

[0004] HER3 ligands include neuregulin 1 (NRG-1) and neuregulin 2 (NRG-2). After ligand binding to HER3, HER3 cannot form homodimers and can only form heterodimers with another receptor to induce intracellular phosphorylation. Furthermore, because HER3 has almost no intracellular tyrosine kinase activity and cannot initiate downstream signaling pathways on its own, HER3 needs to bind to other EGFR family receptors, such as EGFR and HER2, or non-EGFR family receptors, including the MET factor receptor and FGFR2, to form heterodimers to participate in signal transduction and initiate pathways related to cell proliferation or differentiation. HER3 readily forms heterodimers with EGFR and HER2, but has a lower affinity for HER4. Heterodimers formed by HER3 and HER2 can activate the phosphatidylinositol 3-kinase (PI3K) pathway. The activated PI3K / AKT signaling pathway is crucial for cancer cell survival and is the primary cause of cancer cell drug resistance.

[0005] HER3 is highly expressed in a variety of cancer types, including breast cancer, colon cancer, lung cancer, pancreatic cancer, skin cancer, gastric cancer, ovarian cancer, and melanoma, and its high expression is also associated with disease progression and poor prognosis. Compared with other EGFR family members, HER3 overexpression alone is not carcinogenic. However, heterodimers formed by HER3 and HER2 play an important role in the development of various types of cancer. Studies have shown that upregulation of HER3 expression is one of the mechanisms by which tumor cells escape inhibition by EGFR family tyrosine kinases. In addition, signal transduction mediated by HER2 and HER3 heterodimers is associated with resistance to the EGFR tyrosine kinase inhibitor (TKI), gefitinib, in head and neck cancer and breast cancer. In colorectal cancer, increased expression of the HER3 ligand NRG1 and HER2 and HER3 heterodimers can lead to resistance to the EGFR antibody, cetuximab. Therefore, HER3 is considered a popular target for tumor treatment.

[0006] The development of monoclonal antibodies targeting HER3 is expected to become an effective treatment for relapsed tumors that overexpress EGFR and HER2. Because HER3 lacks significant kinase activity, anti-HER3 antibodies primarily block the interaction between HER3 and its ligands or other EGFR family receptors, or recruit immune cells to kill cancer cells. Currently, while some HER3-targeted antibodies are in clinical development and preclinical evaluation, none are marketed. Furthermore, bispecific antibodies and antibody-drug conjugates (ADCs) targeting HER3 are also in clinical trials. Among these, patritumab deruxtecan, an antibody-drug conjugate developed by Daiichi Sankyo, is in Phase III clinical trials. Patritumab deruxtecan combines the anti-human HER3 antibody patritumab with the small molecule drug DXd. Current clinical trials have shown some efficacy in patients with non-small cell lung cancer resistant to EGFR inhibitors. However, as the importance of HER3 in tumor progression and drug resistance becomes increasingly apparent, the development of drugs targeting HER3 is becoming increasingly important.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide an antibody against human epidermal growth factor 3, a preparation method thereof and an application thereof.

[0009] In a first aspect of the present invention, a HER3-specific antibody is provided, wherein the antibody has a light chain variable region and a heavy chain variable region, and

[0010] The amino acid sequence of CDR1 of the light chain variable region is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 12, SEQ ID NO: 18, and SEQ ID NO: 27;

[0011] The amino acid sequence of CDR2 of the light chain variable region is selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 31, SEQ ID NO: 13, and SEQ ID NO: 28;

[0012] The amino acid sequences of CDR3 of the light chain variable region are: SEQ ID NO: 23, SEQ ID NO: 32, SEQ ID NO: 14, SEQ ID NO: 19;

[0013] The amino acid sequence of CDR1 of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 15;

[0014] The amino acid sequence of CDR2 of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 34, and SEQ ID NO: 16;

[0015] The amino acid sequence of CDR3 of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 29.

[0016] In one or more embodiments, the antibody is antibody (a) (which may include humanized (h) HE4 / murine (m) HE4 / chimeric (c) HE4, etc.), and the amino acid sequences of its light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; the amino acid sequences of its heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0017] In one or more embodiments, the antibody is antibody (c) (which may include humanized HE6 / mouse HE6 / chimeric HE6, etc.), and the amino acid sequences of its light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively; the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively.

[0018] In one or more embodiments, the antibody is antibody (d) (which may include humanized HE1 / mouse HE1 / chimeric HE1, etc.), and the amino acid sequences of its light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; the amino acid sequences of its heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0019] In one or more embodiments, the antibody is antibody (e) (which may include humanized HE2 / murine HE2 / chimeric HE2, etc.), and the amino acid sequences of its light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 18, SEQ ID NO: 13 and SEQ ID NO: 19, respectively; the amino acid sequences of its heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 20, respectively.

[0020] In one or more embodiments, the antibody is antibody (f) (which may include humanized HE5 / mouse HE5 / chimeric HE5, etc.), and the amino acid sequences of its light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 19, respectively; the amino acid sequences of its heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 29, respectively.

[0021] In one or more embodiments, the antibody includes: a monoclonal antibody, a single-chain antibody (scFv), a domain antibody, a Fab fragment, a Fd fragment, a Fv fragment, and a F(ab')2 fragment.

[0022] In one or more embodiments, the antibody is a humanized antibody, a chimeric antibody, or a murine antibody.

[0023] In one or more embodiments, the chimeric antibody has the light chain variable region and heavy chain variable region of a murine antibody, but the heavy chain constant region of the antibody is replaced by the human antibody IgG1 heavy chain constant region, and the light chain constant region of the antibody is replaced by the human kappa light chain constant region.

[0024] In one or more embodiments, the humanized antibody is formed by retaining the murine CDR region in the antibody variable region, but replacing the FR region sequences of the heavy and light chain variable regions with sequences that most closely resemble the FR regions of human antibody germline genes, based on the murine variable region sequence. Preferably, the heavy chain constant region of the humanized antibody is a human IgG1 heavy chain constant region, and the light chain constant region is a human kappa light chain constant region.

[0025] In one or more embodiments, the light chain FR region of the humanized antibody has: FR1 shown at positions 1-23, FR2 shown at positions 35-49, FR3 shown at positions 57-88, and FR4 shown at positions 98-108 of SEQ ID NO: 36.

[0026] In one or more embodiments, the heavy chain FR region of the humanized antibody has: FR1 shown at positions 1-27, FR2 shown at positions 36-47, FR3 shown at positions 60-96, and FR4 shown at positions 112-122 of SEQ ID NO: 37.

[0027] In one or more embodiments, the antibody is an antibody (humanized antibody) having a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 36 and a heavy chain variable region as shown in SEQ ID NO: 37.

[0028] In one or more embodiments, the antibody is an antibody (mHE4) having a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 5 and a heavy chain variable region as shown in SEQ ID NO: 6.

[0029] In one or more embodiments, the antibody is an antibody (mHE6) having an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 9 and a heavy chain variable region as shown in SEQ ID NO: 10.

[0030] In one or more embodiments, the antibody is an antibody (mHE7) having an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 9 and a heavy chain variable region as shown in SEQ ID NO: 11.

[0031] In one or more embodiments, the antibody is an antibody (mHE1) having an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 1 and a heavy chain variable region as shown in SEQ ID NO: 2.

[0032] In one or more embodiments, the antibody is an antibody (mHE2) having an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 3 and a heavy chain variable region as shown in SEQ ID NO: 4.

[0033] In one or more embodiments, the antibody is an antibody (mHE5) having an amino acid sequence as shown in SEQ ID NO: 7 and a heavy chain variable region as shown in SEQ ID NO: 8.

[0034] In one or more embodiments, the antibody further includes: an antibody whose heavy chain variable region amino acid sequence is more than 85% (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identical to any of the above-mentioned heavy chain variable region amino acid sequences, and an antibody whose light chain variable region amino acid sequence is more than 85% (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identical to any of the above-mentioned light chain variable region amino acid sequences, and which has the same function as the antibody described in the embodiments of the present invention.

[0035] In another aspect of the present invention, a polynucleotide encoding the antibody is provided.

[0036] In another aspect of the present invention, a construct (such as an expression vector) is provided, which comprises the polynucleotide.

[0037] In another aspect of the present invention, an antibody expression system is provided, wherein the expression system contains the construct or the exogenous polynucleotide is integrated into the genome.

[0038] In one or more embodiments, the antibody expression system is a cell expression system.

[0039] In another aspect of the present invention, a method for preparing any of the above-mentioned HER3-specific antibodies is provided, comprising: expressing the antibody using the antibody expression system under conditions suitable for expressing the antibody, thereby obtaining the antibody.

[0040] In one or more embodiments, the preparation method further comprises: purifying and isolating the antibody.

[0041] In another aspect of the present invention, the use of any of the above-described antibodies is provided for: preparing an anti-tumor drug that specifically targets tumor cells expressing HER3; preparing an antibody-drug conjugate or immunoconjugate; preparing a bifunctional or multifunctional antibody; preparing a reagent for diagnosing a tumor that expresses HER3; or preparing an immune cell modified with a chimeric antigen receptor.

[0042] In another aspect of the present invention, an immunoconjugate is provided, comprising any of the antibodies described above; and a functional molecule linked thereto (including covalently linked, coupled, attached, and adsorbed).

[0043] In one or more embodiments, the functional molecules include (but are not limited to): tumor-suppressing molecules, molecules targeting tumor surface markers, cytotoxins, radioactive isotopes, biologically active proteins, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.

[0044] In one or more embodiments, the tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine.

[0045] In one or more embodiments, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.

[0046] In one or more embodiments, the molecule targeting a surface marker of an immune cell is an antibody or a ligand that binds to a surface marker of an immune cell.

[0047] In one or more embodiments, the anti-tumor toxins include (but are not limited to): toxins that act on tubulin; toxins or derivatives thereof that act on DNA; compounds or derivatives thereof that act on intracellular metabolism, transcription, translation or signal transduction.

[0048] In one or more embodiments, the anti-tumor cytokines include (but are not limited to): IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof.

[0049] In one or more embodiments, the antibody that binds to a tumor surface marker is an antibody that recognizes antigens other than HER3, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16.

[0050] In one or more embodiments, the antibody that binds to an immune cell surface marker, the antigen 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.

[0051] In one or more embodiments, the toxins acting on tubulin include: monomethyl auristatin (including its derivatives), maytansinoid (including its derivatives).

[0052] In one or more embodiments, the toxins acting on DNA include: DXd (including its derivatives), Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines (PBDs), SN-38 (including its derivatives).

[0053] In one or more embodiments, the toxin also includes: related compounds and their derivatives that act on other functions in tumor cells such as metabolism, transcription, translation, signal transduction, etc.

[0054] In one or more embodiments, the anti-tumor toxin is linked to the antibody via a linker, which includes but is not limited to: maleimide-GGFG peptide linker, mc-Val-Cit-PAB (maleimidocaproyl valine-citrulline para-amino benzyloxycarbonyl), or mc-Val-Ala-PAB (maleimidocaproyl valine-alanine para-amino benzyloxycarbonyl).

[0055] In one or more embodiments, the detectable markers include (but are not limited to): fluorescent markers and colorimetric markers.

[0056] In one or more embodiments, the intracellular signaling region includes (but is not limited to): CD3ζ chain, FcεRIγ tyrosine activation motif, and intracellular signaling regions of co-stimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, and CD40.

[0057] In one or more embodiments, the transmembrane region includes (but is not limited to): the transmembrane region of CD8 or CD28.

[0058] In another aspect of the present invention, a pharmaceutical composition (including a diagnostic composition, such as a diagnostic reagent) is provided, comprising: any of the aforementioned antibodies or any of the aforementioned immunoconjugates.

[0059] In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0060] In another aspect of the present invention, there is provided use of the aforementioned antibody, immunoconjugate or pharmaceutical composition in the preparation of a preparation, a kit or a medicine box for diagnosing or treating a tumor; the tumor is a tumor expressing HER3.

[0061] 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.

[0062] In another aspect of the present invention, a kit or a pharmaceutical kit is provided, comprising the antibody, the immunoconjugate or the pharmaceutical composition.

[0063] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG1 shows the results of ELISA and FACS testing of serum titers in immunized mice according to the present invention.

[0065] FIG2 shows the ELISA test results of the chimeric antibody of the present invention against human and monkey HER3.

[0066] FIG3 shows the FACS detection results of the chimeric antibody of the present invention on human and monkey HER3.

[0067] FIG4 shows the results of an antigen binding epitope experiment between the chimeric antibody of the present invention and patritumab.

[0068] FIG5 is the detection result of endocytosis of the chimeric antibody of the present invention.

[0069] FIG6 shows the killing function of the chimeric antibody-drug conjugate of the present invention on tumor cells.

[0070] FIG7 shows the ELISA detection results of the humanized antibody of the present invention against human and monkey HER3.

[0071] FIG8 is the FACS detection result of the humanized antibody of the present invention.

[0072] FIG9 shows the binding of the humanized version of the present invention and patritumab to different subdomains of HER3.

[0073] FIG10 shows the binding ability of the humanized antibody of the present invention to EGFR, HER2 and HER4.

[0074] FIG11 is a cell imaging detection result of endocytosis of the humanized antibody of the present invention.

[0075] FIG12 is the FACS detection result of the endocytosis of the humanized antibody of the present invention.

[0076] FIG13 shows the tumor-killing function of the humanized antibody-drug conjugate of the present invention.

[0077] FIG14 shows the anti-tumor effect of the antibody-drug conjugate of the present invention on SW620 tumor-bearing mice.

[0078] FIG15 shows the anti-tumor effect of the antibody-drug conjugate of the present invention on DiFi tumor-bearing mice.

[0079] FIG16 shows the process from hybridoma preparation to obtaining humanized antibodies. DETAILED DESCRIPTION

[0080] After in-depth research, the present inventors have revealed a HER3-specific antibody that can specifically bind to HER3 and be internalized by cells, and can be used to inhibit diseases related to HER3 expression (including overexpression) or affected by HER3 function, such as tumors.

[0081] the term

[0082] As used herein, the term "antibody" or "immunoglobulin" is used as a general term herein, including full-length antibodies, single-chain antibodies, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments). In addition, the term "sequence" used herein (for example, in terms such as "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0083] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0084] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from a non-human species immunoglobulin, wherein the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only complementarity determining regions (CDRs) transplanted into appropriate framework regions in the variable domain. The antigen binding site may be wild type or modified by one or more amino acid substitutions, for example, to be more similar to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences. Other forms have one or more CDRs that have been altered relative to the original antibody.

[0085] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity.

[0086] An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

[0087] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0088] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0089] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0090] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0091] The term "treatment / prevention" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the condition.

[0092] The term "detectable label" refers to a marker that can be attached to an antibody and used to determine the presence and amount of a specific target in a subject to be detected. The "detectable label" may include, but is not limited to, enzymes, fluorescent markers, radionuclides, quantum dots, colloidal gold, and the like. More specifically, it may be selected from, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0093] In the present invention, the tumor is a HER3 tumor; preferably, the tumor 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.

[0094] Antibody

[0095] The present invention provides an anti-HER3 antibody, wherein the anti-HER3 antibody has 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 having an amino acid sequence of a heavy chain variable region that is 85% or more (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequences listed in Table 6 or Table 2, and an antibody having an amino acid sequence of a light chain variable region that is 85% or more (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequences listed in Table 6 or Table 2, and an antibody having the same function or similar activity as the antibodies described in the embodiments of the present invention.

[0096] The antigen-binding properties of an antibody are typically determined by three complementary determining regions (CDRs). These CDRs are arranged in a sequence with the FRs, which do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheets formed by the intervening FRs, forming the antibody's antigen-binding site. The CDRs represent sequences of proteins of immunological interest. The antibodies of the present invention include sequence modifications to both the CDRs and the framework regions.

[0097] The anti-HER3 antibodies provided by the present invention can efficiently and specifically bind to human HER3 or monkey HER3, effectively blocking the effects of HER3 and efficiently carrying other substances coupled to them to target cells. After binding to HER3 on the cell surface, they can be rapidly internalized into the cell.

[0098] The present invention also includes an anti-HER3 antibody fusion protein comprising a first domain of the antibody of the present invention and a second domain for extending in vivo half-life and / or binding to effector cells. The fusion protein can be a binding molecule that specifically binds to cells expressing HER3.

[0099] In the second domain, the fragment for extending the half-life in vivo may include serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin (such as an antibody against serum albumin), a polyethylene glycol-liposome complex, etc. In the second domain, the fragment that has a binding effect on effector cells may include an immunoglobulin Fc region, etc., preferably selected from the human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations for changing the effector function mediated by Fc, and the effector function includes a combination of one or more of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG may be specifically selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the antibody fusion protein can cause the fusion protein to form a dimer, while extending the in vivo half-life of the fusion protein and increasing Fc-mediated related activities. In one embodiment of the present invention, the immunoglobulin Fc region can be the Fc region of human IgG1, more specifically, it can be a wild-type IgG1 Fc sequence, and mutations that alter Fc-mediated effector functions can be introduced into the sequence, for example, a) mutations that alter Fc-mediated CDC activity; b) mutations that alter Fc-mediated ADCC activity; or c) mutations that alter Fc-mediated ADCP activity. Such mutations are described in 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.

[0100] In the anti-HER3 antibody fusion protein provided by the present invention, a connecting peptide may be provided between the first domain and the second domain. The connecting peptide may be a flexible polypeptide chain composed of alanine (A) and / or serine (S) and / or glycine (G). The length of the connecting peptide may be 3 to 30 amino acids, preferably 3 to 9, 9 to 12, 12 to 16, or 16 to 20 amino acids. In another specific embodiment of the present invention, the length of the connecting peptide may be 8 or 15 amino acids.

[0101] The present invention also provides an isolated polynucleotide encoding the antibody of the present invention or the fusion protein. The polynucleotide may be RNA, DNA, or cDNA. Methods for providing the isolated polynucleotide should be known to those skilled in the art. For example, the isolated polynucleotide may be prepared by automated DNA synthesis and / or recombinant DNA technology, or may be isolated from a suitable natural source.

[0102] Constructs and antibody expression systems

[0103] The present invention also provides a construct comprising the isolated polynucleotide of the present invention. Methods for constructing the construct should be known to those skilled in the art. For example, the construct can be constructed by in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, or the like. More specifically, the construct can be constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector herein generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. The vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, and the regulatory sequences may include a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence for the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequences may also include a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice can be used in the present invention.

[0104] Generally speaking, suitable vectors can contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers. For example, these promoters can include, but are not limited to, the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, and can include, for example, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector can also include an enhancer sequence. Insertion of an enhancer sequence in the vector enhances transcription. Enhancers are cis-acting DNA elements, typically about 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0105] The present invention also provides an antibody expression system, comprising a construct of the present invention or an exogenous polynucleotide of the present invention integrated into its genome. Any cell suitable for expression by an expression vector can be used as a host cell. For example, the host cell can 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, specifically including but not limited to Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; or 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 for example, can include but are not limited to microinjection, gene guns, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, or a combination of one or more of the following.

[0106] Immunoconjugates

[0107] The present invention also provides an immunoconjugate comprising the antibody of the present invention or the fusion protein of the present invention. The immunoconjugate generally further comprises a functional molecule linked (including but not limited to covalently linked, coupled, attached, or adsorbed) to the antibody or fusion protein, wherein the functional molecule may include but is not limited to a detectable marker, a cytotoxin, a radioisotope, a biologically active protein, a molecule targeting a tumor surface marker, a molecule that inhibits tumors, a molecule targeting a surface marker of an immune cell, or a combination thereof.

[0108] The method for preparing the immunoconjugate should be known to those skilled in the art. For example, the antibody and / or fusion protein can be linked to the functional molecule directly or through a spacer of appropriate length. The connection method can be chemical cross-linking or genetic engineering fusion expression to obtain the immunoconjugate.

[0109] For therapeutic purposes, it may be appropriate to combine with therapeutic effector groups such as radioactive groups, i.e., groups composed of radioisotopes or radionuclides (e.g. 3 H. 14 C. 15 N. 33 P. 35 S. 90 Y. 99 Tc, 111 ln、 123 1. 125 1. 131 1. 201 TI, 213 Bi), a toxin or a cytotoxic group such as a cytostatic group or a group consisting of or including the same.

[0110] The immunoconjugate may comprise an antibody or fusion protein of the present invention and a detectable label. Detectable labels include, but are not limited to, fluorescent labels, chromogenic labels, and protein tags; such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. More than one label may also be included. The label used to label the antibody for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnostic technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels for detection / analysis / diagnostic techniques and / or methods known in the art are well known to those skilled in the art.

[0111] The antibody of the present invention or fusion protein can be coupled with a labeling group (labeled polypeptide), which can then be used, for example, for diagnostic purposes. Suitable labeling groups can be selected from radioisotopes (such as those mentioned above) or groups containing radioisotopes, radionuclides, fluorescent groups (such as fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescein and derivatives thereof such as FITC, cyanine dyes), enzyme groups (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase), chemiluminescent groups, biotin groups, metal particles (such as gold particles), magnetic particles (such as having a core containing magnetite (Fe3O4) and / or maghemite (Fe2O3)), predetermined polypeptide groups, etc.

[0112] The immunoconjugate may comprise an antibody or fusion protein of the present invention and a molecule that targets a surface marker of an immune cell. The molecule that targets a surface marker of an immune cell can recognize immune cells and carry the antibody of the present invention to the immune cells. The antibody of the present invention can simultaneously target the immune cells to tumor cells, thereby utilizing the killing effect of the antibody of the present invention while also inducing the immune cells to specifically kill the tumor.

[0113] The immunoconjugate may comprise an antibody or fusion protein of the present invention and at least one molecule that targets a tumor surface marker or a tumor-suppressing molecule. The tumor-suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin. For example, the cytokine may include, but is not limited to, IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, etc. The molecule that targets a tumor surface marker may, for example, act synergistically with the antibody of the present invention to more precisely target tumor cells.

[0114] The immunoconjugate can also be a chimeric antigen receptor (CAR), which can be expressed in immune cells. The “immune cells” and “immune effector cells” can be used interchangeably, including: T lymphocytes, NK cells or NKT cells, etc., preferably, NK cells and T lymphocytes. The chimeric antigen receptor generally comprises an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises the antibody or fusion protein of the present invention. The design of the transmembrane region and the intracellular signaling region based on CAR technology is well known in the art: for example, the transmembrane region adopts the transmembrane region of molecules such as CD8 and CD28, and the intracellular signaling region adopts the intracellular signaling region of the immunoreceptor tyrosine activation motif (ITAM) CD3ζ chain or FcεRIγ tyrosine activation continuation and co-stimulatory signal molecules CD28, CD27, CD137, CD134, MyD88, CD40, etc. More specifically, for T lymphocytes, the first generation of CAR T lymphocytes contains only ITAM in the intracellular signaling region, in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-ITAM, which can stimulate anti-tumor cytotoxic effects; the second generation of CAR T lymphocytes adds the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM- / CD137-ITAM; the B7 / CD28 or 4-1BBL / CD137 co-stimulation effect in the intracellular signaling region causes the continuous proliferation of T lymphocytes, and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while improving the survival cycle and anti-tumor effect of CAR T in the body; the third generation of CAR T lymphocytes, in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improving the survival cycle of CAR T in the body and its anti-tumor effect.

[0115] According to the above, the chimeric antigen receptor prepared using the antibody or fusion protein of the present invention can be a chimeric antigen receptor comprising the following sequentially connected extracellular binding regions, transmembrane regions and intracellular signaling regions: 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 (CD28a) of the CD28 molecule, the intracellular signaling region (CD28b) of the CD28 molecule 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.

[0116] The chimeric antigen receptor is expressed on the surface of immune effector cells, and while utilizing the killing effect of the antibody of the present invention, the immune effector cells can also have a highly specific cytotoxic effect on tumor cells expressing HER3.

[0117] As a preferred embodiment of the present invention, the antibodies of the present invention can be linked to tumor-suppressing molecules. Preferably, the tumor-suppressing molecules are anti-tumor toxins, including toxins that act on DNA, such as DXd, duocarmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), SN-38, and related compounds and their derivatives; toxins that act 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 analogs, isomers, and precursors of these small molecule toxins.

[0118] Pharmaceutical compositions and kits

[0119] The present invention also provides a pharmaceutical composition comprising the anti-HER3 antibody of the present invention, or the fusion protein of the anti-HER3 antibody of the present invention, or the immunoconjugate of the present invention.

[0120] The pharmaceutical composition may also include various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may include but are not limited to: buffers such as acetate, Tris, phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parahydroxybenzoates such as methyl parahydroxybenzoate or propyl parahydroxybenzoate; catechol; cyclohexanol; 3-pentanol; and cresol); proteins such as blood albumin, gelatin or immunoglobulin; 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 (such as Zn-protein complexes); and / or nonionic surfactants such as Or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile. Methods for achieving sterility of pharmaceutical preparations should be known to those skilled in the art. For example, it can be achieved by filtration through a sterile filtration membrane. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the desired dosage form of the pharmaceutical composition to prepare it into different dosage forms. For example, the pharmaceutical composition of the present invention can be in various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0121] In the pharmaceutical composition, the content of the fusion protein and the immunoconjugate is generally an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the subject to be treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, the content of the fusion protein and the immunoconjugate can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%.

[0122] The fusion proteins, immunoconjugates, and pharmaceutical compositions of the present invention can be administered as a single active ingredient or in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include the fusion protein, immunoconjugate, or pharmaceutical composition in combination with at least one other anti-tumor drug. As another example, the combination therapy can include the fusion protein, immunoconjugate, or pharmaceutical composition in combination with antibodies targeting other tumor-specific antigens, such as, but not limited to, anti-EGFR antibodies, anti-VEGF antibodies, anti-HER2 antibodies, or anti-Claudin18A2 antibodies. The inhibitor can preferably be a monoclonal antibody.

[0123] The present invention also provides a detection kit comprising the antibody, fusion protein, or immunoconjugate described herein. The kit may further include, as needed, a container, controls (negative or positive controls), a buffer, adjuvants, etc., which can be selected by those skilled in the art based on specific circumstances. The kit may also include instructions for use to facilitate operation by those skilled in the art.

[0124] The present invention further provides a method for detecting HER3 protein using the antibody, including but not limited to qualitative detection, quantitative detection and localization detection. Specifically, the detection method includes but is not limited to immunofluorescence assay, immunohistochemistry and radioimmunoassay.

[0125] A method for detecting the presence of HER3 protein in a sample may include: contacting the sample with an antibody of the present invention; observing whether an antibody complex forms; the formation of an antibody complex indicates the presence of HER3 protein in the sample. The sample may be a cell and / or tissue sample; the sample may be fixed or dissolved in a medium; and the level of HER3 protein in the fixed or dissolved sample is detected. In some embodiments, the test subject may be a cell-containing sample in a cell preservation solution. In other embodiments, the antibody is further conjugated with a fluorescent dye, chemical substance, peptide, enzyme, isotope, label, or the like that can be detected or detected by other reagents.

[0126] use

[0127] The present invention also provides use of the antibody, fusion protein, immunoconjugate or pharmaceutical composition of the present invention in the preparation of a pharmaceutical composition for diagnosing, treating or preventing a disease associated with cells expressing (or overexpressing) HER3.

[0128] The "therapeutically effective amount" of the fusion protein, immunoconjugate, 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 of the disease, or prevents damage or disability caused by the pain of the disease. For example, for the treatment of HER3-related tumors (including ovarian cancer, endometrial cancer, breast cancer, cervical cancer, etc.), a "therapeutically effective amount" preferably inhibits cell growth 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% relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by tests known to those skilled in the art. A therapeutically effective amount of the fusion protein, immunoconjugate, or pharmaceutical composition is generally capable of reducing tumor size or otherwise alleviating symptoms in the subject. Those skilled in the art can select an appropriate therapeutically effective amount based on the actual situation, for example, 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., determination of dosage, etc.) can be determined by a physician, typically taking into account factors including, but not limited to, the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors. A prophylactically effective amount refers to an amount effective to achieve the desired prophylactic effect at the necessary dosage and time. Typically, but not necessarily, a prophylactic dose is administered to a subject before the onset of the disease or in the early stages of the disease, so a "prophylactically effective amount" is generally lower than a "therapeutically effective amount."

[0129] The beneficial technical effects of the present invention include:

[0130] Currently, there are no anti-HER3 monoclonal antibodies on the market in this field, and the efficacy of anti-HER3 monoclonal antibodies is not significant based on the results of current clinical trials. The anti-HER3 antibodies of the present invention have high affinity and ideal antigen binding activity.

[0131] The anti-HER3 antibodies of the present invention can be rapidly and effectively internalized by cells and can be prepared into ADC drugs or bispecific antibodies to play a role in tumor treatment. Obviously, bispecific antibodies or antibody-drug conjugates (ADCs) targeting HER3 have greater prospects.

[0132] Compared with the control antibody patritumab, the antibodies of the present invention, such as hHE4-G1W, bind to different epitopes of HER3, thus providing a new targeting antibody for clinical use.

[0133] Compared with the control antibody patritumab, hHE4-G1W has a higher affinity for HER3-expressing tumor cells and a faster internalization rate. After being coupled with small molecules, it can target and bind to HER3-expressing tumor cells with high affinity, resulting in the best cell killing effect.

[0134] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0135] Example 1. Preparation of animals for hybridoma fusion

[0136] Several SJL mice (numbered 10886, 10888, 10889, 10890, 10891, 10892, 10893, 10894, and 10895) were immunized intraperitoneally with a His-tagged fusion protein of the extracellular portion of human HER3 (hHER3-ECD-His) as an immunogen to stimulate the production of anti-human HER3 antibodies in the mice. Serum titers were determined by ELISA. The hHER3-ECD-His protein was adsorbed onto a 96-well microtiter plate, followed by the addition of serially diluted serum, incubation at room temperature for 1 hour, and plate washing. HRP-conjugated rabbit anti-mouse full-length secondary antibody (Thermo Fisher) was then added, incubation at room temperature for 1 hour, and plate washing. Finally, TMB was used for color development, and absorbance at 450 nm was recorded using a microplate reader. As shown in Figure 1 (A) and (B), the titers of mouse sera at the protein level were all above 1:1000K.

[0137] Next, A375 cells expressing HER3 were used to detect serum titers using FACS. First, the A375 cells were digested with trypsin and collected by centrifugation. The cells were resuspended in PBS and counted. The cells were added to a 96-well plate and centrifuged to remove the supernatant. The serum from each group of immunized mice was then added. After incubation at room temperature for 30 minutes, the cells were washed twice. A PE-labeled goat anti-mouse full-length secondary antibody (Jackson) was added. After incubation in the dark for 30 minutes, the cells were washed once. The cells were resuspended in PBS and detected by flow cytometry. The results are shown in Figures C and D of Figure 1. At the cellular level, the serum from the 10890 mouse had the highest titer, reaching 182K. The titers of the other mice all reached 1:20K or higher, indicating that the antibodies produced in the mouse serum can specifically recognize HER3 expressed on the surface of A375 cells.

[0138] The results of serum titer detection showed that anti-human HER3 antibodies were produced by immunizing mice with human HER3 extracellular domain protein. Based on the titer results, 10889 and 10890 mice were selected for hybridoma fusion.

[0139] Example 2: Obtaining antibodies with higher affinity

[0140] Splenic lymphocytes from immunized mice were fused with SP2 / 0 myeloma cells using electrofusion to generate hybridoma cells that secrete specific anti-human HER3 antibodies and are capable of indefinite proliferation. Subclonal screening of the hybridomas yielded multiple anti-human HER3-specific antibodies. ELISA and FACS analysis of these subclones was performed using the same methods as in Example 1, resulting in the identification of five antibodies with high affinity. Using either human HER3 or cyno HER3 as the antigen, the experimental results are shown in Table 1.

[0141] Table 1

[0142] From the results, it can be seen that the five subclones can specifically bind to human and monkey HER3 antigens and have good binding effects.

[0143] Example 3. Antibody Sequences

[0144] 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 genes were amplified from the reverse-transcribed cDNA using PCR, ligated into the pMDTM18-T vector (TAKARA), and transformed into competent DH5α cells. Sequencing yielded the heavy and light chains of the monoclonal antibodies. Tables 2 and 3 respectively show the variable region amino acid sequences and CDR amino acid sequences of the mouse monoclonal antibody against human HER3. Sequence analysis revealed that amino acids 73-75 ("NKS") in the mHE6 heavy chain variable region were glycosylation sites. Therefore, when constructing the chimeric antibody, the "N" at position 73 was mutated to a "D," resulting in the designation of the mHE7 heavy chain. The mHE7 light chain variable region is identical to that of the mHE6 light chain.

[0145] Table 2

[0146] Table 3

[0147] Example 4. Expression of chimeric antibodies

[0148] Using PrimeSTAR@Max DNA Polymerase (TAKARA), the antibody heavy and light chain variable region nucleic acid genes obtained in Example 3 were PCR cloned and then combined with the nucleic acid genes encoding the human antibody IgG1 heavy chain constant region (GenBank accession number: AXN93653.1 for its amino acid sequence) and the human kappa light chain constant region (GenBank accession number: AWK57456.1 for its amino acid sequence) to form full-length heavy and light chain genes. The target genes were separated by agarose gel electrophoresis and obtained using a gel recovery kit (Omega). Finally, the target genes were ligated into the expression vector using the ClonExpress Ultra One Step Cloning Kit (Vazyme).

[0149] After sequencing verified the correctness of the expression vector, chimeric antibodies were expressed, corresponding to the heavy and light chains of mHE1-mHE6, to obtain chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE6-G1W, and cHE7-G1W, respectively. The function of the chimeric antibodies was verified.

[0150] Example 5. Analysis of the binding ability of chimeric protein to antigen (ELISA)

[0151] ELISA was used to examine the binding ability of chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE6-G1W, and cHE7-G1W to the extracellular domain of human and monkey HER3 proteins. Human HER3-ECD-His and monkey HER3-ECD-His (Sino Bio) proteins were adsorbed onto ELISA plates. The plates were incubated with 5% skim milk powder at room temperature for 1 hour, then washed. Serially diluted antibodies were added, incubated at room temperature for 1 hour, and then washed. HRP-labeled goat anti-human Fc secondary antibody (Jackson) was then added, incubated at room temperature for 1 hour, and then washed. Finally, TMB was used for color development, and the absorbance at 450 nm was recorded using a microplate reader.

[0152] As shown in Figure 2, Panel A, all chimeric antibodies bound to human HER3 with similar affinities to the control antibody, patritumab (from patent WO2007077028). However, Figure 2, Panel B, shows that the EC50s for cHE6-G1W and cHE7-G1W binding to monkey HER3 were 3-4 times lower than those for patritumab.

[0153] Example 6. Analysis of the binding ability of chimeric protein to antigen (FACS)

[0154] FACS was used to detect 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. CHO cells overexpressing human HER3 and monkey HER3 were centrifuged separately, resuspended in PBS, counted, added to a 96-well plate, centrifuged, and the supernatant removed. Serially diluted antibodies were added, incubated at 4°C for 30 minutes, and then the cells were washed twice. A secondary antibody with a PE-conjugated goat anti-human IgG Fc tag (Jackson) was added, incubated at 4°C in the dark for 30 minutes, and then the cells were washed once. The cells were resuspended in PBS and detected using a flow cytometer.

[0155] As shown in Figure 3A, it was found that the EC50 of chimeric antibodies cHE1-G1W, cHE2-G1W, cHE4-G1W and cHE5-G1W binding to human HER3 was 2- to 7-fold higher than that of patritumab, and cHE6-G1W and cHE7-G1W had similar affinity to patritumab.

[0156] As shown in Figure 3B, the EC50 value of cHE4-G1W binding to monkey HER3 was 2-fold higher than that of patritumab, while the affinity of other chimeric antibodies binding to monkey HER3 was significantly reduced.

[0157] Example 7. Affinity analysis of chimeric antibodies

[0158] Octet was used to test the affinity of the six chimeric antibodies to human HER3-ECD-His and monkey HER3-ECD-His proteins. The results are shown in Table 4.

[0159] Judging from the Ka values, the binding rates of the six chimeric antibodies to human HER3-ECD-His (hHER3-ECD-His) and monkey HER3-ECD-His (cyno HER3-ECD-His) proteins were better than those of patritumab.

[0160] Based on Kd values, cHE4-G1W dissociates more slowly than patritumab from both hHER3-ECD-His and cyno HER3-ECD-His proteins. In contrast, cHE6-G1W and cHE7-G1W dissociate more slowly from hHER3-ECD-His than patritumab, but dissociate more rapidly from cyno HER3-ECD-His proteins.

[0161] Judging from the KD values, the affinities of cHE4-G1W, cHE6-G1W, and cHE7-G1W for hHER3-ECD-His were better than those of patritumab. Moreover, cHE6-G1W and cHE7-G1W had similar affinities for both hHER3-ECD-His and cyno HER3-ECD-His proteins, indicating that removal of the glycosylation site of cHE6-G1W did not affect its affinity for hHER3-ECD-His and cyno HER3-ECD-His.

[0162] Table 4

[0163] Example 8. Analysis of thermal stability of chimeric antibodies

[0164] The thermal stability of six chimeric antibodies and patritumab was tested using dynamic light scattering (DLS). The DLS (Wyatt) instrument detects the light scattering of the antibody when heated from 25°C to 85°C, thereby obtaining the polymerization temperature T of the antibody. agg value.

[0165] The results are shown in Table 5. Through testing, it was found that the polymerization temperature T agg The values ​​of are all greater than 60℃. Among them, the Tagg The values ​​are all greater than those of the control antibody patritumab, indicating that the thermal stability of these four chimeric antibodies is better than that of patritumab.

[0166] Table 5

[0167] Example 9. Analysis of chimeric antibody binding epitopes

[0168] Octet was used to further analyze the human HER3 epitopes bound by cHE1-G1W, cHE2-G1W, cHE4-G1W, cHE5-G1W, cHE7-G1W, and patritumab. The AMC sensor was first bound to 10 μg / mL of mouse Fc-tagged human HER3-ECD protein. The first antibody was then bound until the signal reached saturation. Finally, the second antibody was added to observe whether the signal increased. If the signal increased, the first and second antibodies bound to different epitopes on human HER3. If the signal did not change, the first and second antibodies bound to the same epitope on human HER3.

[0169] The results obtained by this method are shown in Figure 4. cHE1-G1W, cHE2-G1W, cHE4-G1W and cHE5-G1W bind to the same epitope, and cHE7-G1W binds to a different epitope. These antibodies do not compete with patritumab for binding to human HER3, indicating that these antibodies and patritumab bind to different sites on human HER3.

[0170] Example 10: Analysis of cellular endocytosis after chimeric antibody binding to antigen

[0171] To test whether cHE4-G1W, cHE5-G1W, and cHE7-G1W in the present invention can be internalized into cells together with human HER3 after binding to human HER3, 10 nM antibodies were mixed with A375 cells overexpressing human HER3, incubated at 4°C for 1 hour, and then unbound antibodies were washed away. The 0-hour sample was placed at 4°C, and then the cells were placed in a 37°C incubator for incubation. After 1 hour, 2 hours, and 3 hours, PE-labeled goat anti-human Fc secondary antibody (Jackson) was added to the sample, incubated at 4°C for 30 minutes, and then unbound secondary antibody was washed away. Finally, flow cytometry was used for detection.

[0172] The analysis results are shown in Figure 5. After binding to HER3 on the surface of A375 cells, cHE4-G1W, cHE5-G1, and cHE7-G1W were all internalized by the cells. The internalization rate of cHE7-G1W was close to that of the control antibody patritumab.

[0173] Example 11. Preparation of chimeric antibody-based conjugate drugs and analysis of their tumor killing effects

[0174] Three chimeric antibodies, cHE4-G1W, cHE5-G1W, and cHE7-G1W, were prepared into antibody-drug conjugates (ADCs) to test their cytotoxicity against A375 tumor cells overexpressing human HER3 and OVCAR3 tumor cells expressing HER3. The chimeric antibodies were first incubated with TCEP (Sigma) to break the interchain disulfide bonds. Maleimide-GGFG-DXd (MedChemExpress) was then added for conjugation. Finally, the antibodies were purified using a desalting column to remove excess small molecule drug. The resulting ADCs were named cHE4-G1W-DXd, cHE5-G1W-DXd, cHE7-G1W-DXd, and patritumab-DXd, respectively. IgG-DXd was used as a negative control. By hydrophobic interaction chromatography analysis, the ratio of cHE4 antibody to DXd was 1:7, the ratio of cHE5 antibody to DXd was 1:6.9, the ratio of cHE7 antibody to DXd was 1:7.7, the ratio of patritumab antibody to DXd was 1:8, and the ratio of IgG to DXd was 1:7.5.

[0175] A375 and OVCAR3 cells, which overexpress human HER3, were seeded at 5,000 cells per well in 96-well plates. Antibody-drug conjugates cHE4-G1W-DXd, cHE5-G1W-DXd, cHE7-G1W-DXd, patritumab-DXd, and the negative control IgG-DXd were serially diluted and added to the 96-well plates containing HER3-overexpressing A375 and OVCAR3 cells, respectively. The cells were cultured at 37°C for 5 days. Cell viability was assessed using CellTiter-Glo (Promega) to assess the cell-killing effect of the antibody-drug conjugates.

[0176] The results, shown in Figure 6A and B, show that cHE4-G1W-DXd, cHE5-G1W-DXd, and cHE7-G1W-DXd all had significant cytotoxic effects on A375 cells overexpressing human HER3, with cHE4-G1W-DXd having a 2-fold greater cytotoxic effect than the control antibody patritumab-DXd. On OVCAR3 cells, cHE4-G1W-DXd, cHE5-G1W-DXd, and cHE7-G1W-DXd also had cytotoxic effects compared to the negative control IgG-DXd, with similar cytotoxic effects to patritumab-DXd.

[0177] Example 12: Preparation of humanized antibodies

[0178] Based on the experimental results from Examples 5 to 11, the chimeric antibody cHE4-G1W was selected for humanization. The variable regions of the light and heavy chain sequences of the cHE4-G1W antibody were analyzed using the IMGT database. The CDR1, CDR2, and CDR3 regions, which are crucial for antigen binding, are shown in Table 3. During humanization, the CDR regions were retained, while the murine FR regions were replaced with those that most closely resembled the FR regions of the human antibody germline gene. The resulting humanized antibody has its light and heavy chain variable regions shown in Table 6, respectively.

[0179] Table 6

[0180] The amino acid sequences of the hHE4 antibody's heavy and light chain variable regions were converted to nucleotide sequences and codon-optimized before gene synthesis. These sequences were then ligated with the human IgG1 heavy and light chain constant region genes and constructed into expression vectors. Once sequencing confirmed, the plasmids were extracted and transfected into ExpiCHO cells for expression. Protein A purification was performed to obtain the humanized antibody hHE4-G1W. A schematic diagram of the humanized antibody production process is shown in Figure 16.

[0181] Example 13: Affinity analysis of humanized antibodies

[0182] ELISA was used to examine the affinity of the humanized antibody hHE4-G1W for human HER3 and monkey HER3 compared to the chimeric antibody cHE4-G1W. Human HER3-ECD-His and monkey HER3-ECD-His proteins were adsorbed onto ELISA plates, blocked with 5% skim milk powder, and incubated at room temperature for 1 hour. The plates were washed and then serially diluted with the antibodies. The plates were incubated at room temperature for 1 hour and then washed again. HRP-labeled goat anti-human Fc secondary antibody (Jackson) was then added, incubated at room temperature for 1 hour, and then washed. Finally, TMB was used for color development, and the absorbance at 450 nm was recorded using a microplate reader. As shown in Figures A and B of Figure 7, the humanized hHE4-G1W antibody exhibited comparable binding to human and monkey HER3 as the chimeric antibody cHE4-G1W, and both exhibited superior affinity to the control antibody patritumab.

[0183] Example 14: Analysis of Antigen Binding Ability of Humanized Antibodies

[0184] 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 using FACS technology, using the same method as in Example 6.

[0185] The results are shown in Figure 8. Whether in CHO cells overexpressing human HER3 (Figure A) or original A375 (Figure C), or in CHO cells overexpressing monkey HER3 (Figure B), the humanized hHE4-G1W antibody showed no change in its ability to bind to human and monkey HER3 at the cellular level compared to the chimeric antibody cHE4-G1W. In addition, the EC50 value of hHE4-G1W binding to human HER3 was 4 to 8 times better than that of the control antibody patritumab.

[0186] Example 15: Affinity analysis of humanized antibodies

[0187] Octet was used to test the affinity of the humanized antibody hHE4-G1W for human HER3-ECD-His and monkey HER3-ECD-His proteins. The results are shown in Table 7. Ka, Kd, ​​and KD values ​​indicate that the humanized antibody hHE4-G1W exhibits no significant difference in affinity for both human and monkey proteins compared to the chimeric antibody cHE4-G1W. Furthermore, the KD value of hHE4-G1W is 10-fold stronger than that of patritumab.

[0188] Table 7

[0189] Example 16. Binding epitope analysis of humanized antibodies

[0190] ELISA was used to detect the domain locations of the humanized antibody hHE4-G1W that binds to HER3. According to literature reports (Acta Crystallogr F Struct Biol Commun. 2021 Jul 1; 77(Pt 7): 192-201), the extracellular domain of human HER3 is divided into four domains: domain I (amino acids 56 to 183), domain II (amino acids 184 to 308), domain III (amino acids 309 to 500), and domain IV (amino acids 501 to 643). Fusion proteins of domains I-III (56 to 500) and I-IV (56 to 643) with mFc tags were expressed and purified for ELISA detection. Goat anti-human Fc (Jackson) was adsorbed onto an ELISA plate for 3 hours. After washing, the plate was incubated with 5% skim milk powder at room temperature for 1 hour. After washing, 2 μg / mL hHE4-G1W and patritumab were added and incubated at room temperature for 1 hour. The plate was then washed again. A serial dilution of mFc-tagged HER3 domains I-III (56-500) or HER3 domains I-IV (56-643) fusion proteins was added and incubated for 1 hour. HRP-conjugated rabbit anti-mouse Fc secondary antibody (Thermo Fisher) was then added and incubated at room temperature for 1 hour before washing. Finally, TMB was used for color development, and absorbance at 450 nm was recorded using a microplate reader.

[0191] As shown in Figure 9, the hHE4-G1W antibody can bind to domains I to IV of HER3, but not domains I to III, indicating that the hHE4-G1W antibody binds to domain IV of HER3; while patritumab can bind not only to domains I to IV but also to domains I to III of HER3, indicating that the binding site of the patritumab antibody is on domains I to III.

[0192] Thus, the hHE4-G1W antibody binds to different epitopes on human HER3 than patritumab.

[0193] Example 17: Binding specificity analysis of humanized antibodies

[0194] To demonstrate that hHE4-G1W specifically binds to human HER3, ELISA was used to examine nonspecific binding of hHE4-G1W to human epidermal growth factor receptor (EGFR), HER2, and HER4. Human EGFR-ECD-His, HER2-ECD-His, and HER4-ECD-His proteins were adsorbed onto an ELISA plate and blocked with 5% skim milk powder for 1 hour at room temperature. After washing, the plate was then diluted 3-fold from 25 nM to 0.011 nM. The plate was then incubated for 1 hour at room temperature and washed again. An HRP-conjugated goat anti-human Fc secondary antibody (Jackson) was then added, incubated for 1 hour at room temperature, and then washed. Finally, TMB was used for color development, and the absorbance at 450 nm was recorded using a microplate reader.

[0195] The results are shown in Figures AC in Figure 10 . Neither hHE4-G1W nor patritumab binds to EGFR, HER2, and HER4, indicating that hHE4-G1W only specifically binds to human HER3.

[0196] Example 18: Thermal stability analysis of humanized antibodies

[0197] The thermal stability of the humanized antibody hHE4-G1W was tested using DLS, using the same method as in Example 8. The results, shown in Table 8, show that the thermal stability of hHE4-G1W increased from 69.29°C to 75.54°C after humanization compared to the chimeric antibody cHE4-G1W, demonstrating that the thermal stability of the humanized antibody hHE4-G1W was significantly superior to that of patritumab.

[0198] Table 8

[0199] Example 19: Analysis of cellular endocytosis after humanized antibody binding to antigen

[0200] A cell imaging microplate reader was used to monitor the internalization of hHE4-G1W, hHE4-G1W-DXd, patritumab, and patritumab-DXd after binding to HER3 on the surface of A375 tumor cells. A375 cells overexpressing full-length HER3 were incubated with 100 nM of hHE4-G1W, hHE4-G1W-DXd, patritumab, and patritumab-DXd, respectively, for 30 minutes at 4°C. Unbound antibody was then washed away, and a PE-conjugated goat anti-human Fc secondary antibody (Jackson) was added to the samples. After incubation at 4°C for 30 minutes, unbound secondary antibody was washed away. Samples in which the antibody bound to the A375 cell surface but did not undergo internalization were designated as "0 hour samples." Fluorescence images were taken using a Biotek Cytation 1 Imaging Reader. The cells were then incubated in a 37°C incubator for 3 hours before fluorescence imaging was performed again.

[0201] The results are shown in Figure 11. It can be seen that at 0 hours, there was fluorescence 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 incubation for 3 hours, the fluorescence on the cell surface weakened, and instead the fluorescence accumulated inside the cells, indicating that after 3 hours, hHE4-G1W, hHE4-G1W-DXd, patritumab and patritumab-DXd were internalized from HER3 bound to the surface of A375 cells into the cells.

[0202] Example 20: Analysis of cellular endocytosis after humanized antibody binding to antigen

[0203] The endocytosis rate of humanized antibody hHE4-G1W, chimeric antibody cHE4-G1W4 and control antibody patritumab after binding to A375 cells expressing HER3 on the cell surface was detected by FACS method, with reference to Example 10.

[0204] The results are shown in FIG12 . After hHE4-G1W and cHE4-G1W4 bind to HER3 on the surface of A375 cells, their endocytosis rates are consistent during incubation at 37° C. for 3 hours, and their endocytosis rates are faster than those of the control antibody patritumab.

[0205] Example 21. Preparation of humanized antibody-based conjugate drugs and analysis of their tumor killing effects

[0206] The cytotoxicity of the humanized antibody hHE4-G1W drug conjugate against HER3-expressing HCC1569, A375, and JIMT-1 tumor cells was tested. The antibody and small molecule conjugation method was similar to that described in Example 11. Hydrophobic interaction chromatography analysis revealed that the drug-antibody-to-small molecule ratios for cHE4-G1W-DXd, hHE4-G1W-DXd, patritumab-DXd, and the negative control IgG-DXd were 1:7, 1:8, 1:7.8, and 1:8, respectively.

[0207] The results are shown in Figures AC in Figure 13. hHE4-G1W-DXd, cHE4-G1W-DXd and patritumab-DXd all had significant killing effects on HER3-expressing HCC1569 cells or A375 and JIMT-1 cells overexpressing human HER3, and the killing effect of hHE4-G1W-DXd on JIMT-1 cells overexpressing human HER3 was significantly better than that of patritumab-DXd.

[0208] Example 22: Analysis of the inhibitory effect of humanized antibody-based conjugate drugs on SW620 tumor-bearing animals

[0209] The antitumor efficacy of hHE4-G1W-DXd and cHE5-G1W-DXd in SW620 tumor-bearing mice was analyzed. Tumor-bearing mouse models were established using SCID female mice subcutaneously inoculated with SW620 cells. After model establishment, the mice were randomly divided into nine groups and administered a single dose. Three negative control groups were injected with saline (PBS), a low dose of 3 mg / kg IgG-DXd, and a high dose of 6 mg / kg IgG-DXd. The experimental groups were injected with low doses of 3 mg / kg and 6 mg / kg hHE4-G1W-DXd, cHE5-G1W-DXd, and patritumab-DXd, respectively. The average number of small molecule DXd conjugated to the antibody was eight. Body weight and tumor volume were measured in each group.

[0210] The results in Figure 14 show that compared to the PBS and IgG-DXd control groups, both experimental groups showed inhibitory effects on SW620 tumor growth. However, 3 mg / kg patritumab-DXd showed significant tumor growth 14 days after administration. In contrast, 3 mg / kg hHE4-G1W-DXd showed no tumor growth after administration, and one mouse showed complete tumor regression after 6 mg / kg hHE4-G1W-DXd administration. This suggests that hHE4-G1W-DXd has a superior inhibitory effect on SW620 tumors to patritumab-DXd.

[0211] Example 23: Analysis of the inhibitory effect of humanized antibody-based conjugate drugs on DiFi tumors in tumor-bearing animals

[0212] The antitumor efficacy of hHE4-G1W-DXd and cHE5-G1W-DXd in DiFi tumor-bearing mice was analyzed. A tumor-bearing mouse model was established by subcutaneously inoculating DiFi cells into the back of SCID female mice. After model establishment, the mice were randomly divided into nine groups and dosed once weekly for a total of three times. Three negative control groups were injected with saline (PBS), a low-dose 3 mg / kg IgG-DXd, and a high-dose 6 mg / kg IgG-DXd. The experimental groups were injected with a low-dose 3 mg / kg hHE4-G1W-DXd, cHE5-G1W-DXd, and patritumab-DXd, respectively. The average number of small molecule DXd conjugated to the antibody was eight. Body weight and tumor volume were measured in each group.

[0213] The results in Figure 15 show that compared to the PBS and IgG-DXd control groups, both experimental groups exhibited inhibitory effects on DiFi tumor growth. Patritumab-DXd at 3 mg / kg exhibited relatively poorer tumor inhibition than hHE4-G1W-DXd and cHE5-G1W-DXd at 3 mg / kg. The 6 mg / kg group exhibited similar tumor inhibition effects, indicating that hHE4-G1W-DXd and cHE5-G1W-DXd exhibited comparable or improved inhibitory effects on DiFi tumors compared to patritumab-DXd.

[0214] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

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

2. The antibody according to claim 1, characterized in that The antibodies include: monoclonal antibodies, single-chain antibodies, domain antibodies, Fab fragments, Fd fragments, Fv fragments, F(ab')2 fragments; or 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: An antibody having a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 36, or having 85% or more identity thereto, and a heavy chain variable region as shown in SEQ ID NO: 37, or having 85% or more identity thereto; An antibody having a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 5 or having 85% or more identity thereto, and a heavy chain variable region as shown in SEQ ID NO: 6 or having 85% or more identity thereto; An antibody having a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or having 85% or more identity thereto, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 10, or having 85% or more identity thereto; An antibody having a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, or having 85% or more identity thereto, and a heavy chain variable region as shown in SEQ ID NO: 11, or having 85% or more identity thereto; An antibody having a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 or having 85% or more identity to the sequence, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2 or having 85% or more identity to the sequence; An antibody having a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 or having 85% or more identity thereto, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 4 or having 85% or more identity thereto; An antibody having a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 7 or having 85% or more identity thereto, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 8 or having 85% or more identity thereto. A polynucleotide encoding the antibody according to any one of claims 1 to 3. A construct comprising the polynucleotide of claim 4.

6. An antibody expression system, comprising the construct according to claim 5 or the exogenous polynucleotide according to claim 4 integrated into the genome.

7. A method for preparing the HER3-specific antibody according to any one of claims 1 to 3, comprising: The antibody is expressed using the antibody expression system of claim 6 under conditions suitable for expressing the antibody, thereby obtaining the antibody.

8. Use of the antibody according to any one of claims 1 to 3, for: Preparation of anti-tumor drugs that specifically target tumor cells expressing HER3; preparing antibody drug conjugates or immunoconjugates; Preparation of bifunctional or multifunctional antibodies; preparing a reagent for diagnosing a tumor that expresses HER3; or Preparation of chimeric antigen receptor modified immune cells.

9. An immunoconjugate comprising: The antibody according to any one of claims 1 to 3; and functional molecules connected thereto.

10. The immunoconjugate according to claim 9, characterized in that The functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, cytotoxins, radioactive isotopes, biologically active proteins, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.

11. The immunoconjugate according to claim 10, characterized in that The tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine; or The molecule targeting the tumor surface marker is an antibody or a ligand that binds to the tumor surface marker; or The molecule targeting the surface marker of an immune cell is an antibody or a ligand that binds to the surface marker of an immune cell.

12. The immunoconjugate according to claim 11, characterized in that The anti-tumor toxins include: toxins acting on tubulin; toxins acting on DNA or their derivatives; compounds or their derivatives acting on intracellular metabolism, transcription, translation or signal transduction; or The anti-tumor cytokines include: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof; or The antibody binding to the tumor surface marker is an antibody that recognizes other antigens besides HER3, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16; or The antigens bound by the antibodies binding to immune cell surface markers include: 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 immunoconjugate according to claim 12, characterized in that The toxins acting on tubulin include: monomethyl auristatin, maytansinoids; or The toxins acting on DNA include: DXd, Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines, and SN-38.

14. The immunoconjugate according to claim 11 or 12, characterized in that The anti-tumor toxin is connected to the antibody according to any one of claims 1 to 3 via a linker, and the linker includes: a maleimide-GGFG linker, mc-Val-Cit-PAB, or mc-Val-Ala-PAB.

15. A pharmaceutical composition comprising: The antibody of any one of claims 1 to 3 or the immunoconjugate of any one of claims 9 to 14.

16. Use of the antibody according to any one of claims 1 to 3, the immunoconjugate according to any one of claims 9 to 14, or the pharmaceutical composition according to claim 15 in the preparation of a preparation, a kit or a medicine box for diagnosing or treating a tumor; the tumor is a tumor expressing HER3.

17. The use according to claim 16, characterized in that The tumors include: breast cancer, colon cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer or ovarian cancer.

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

19. A kit or a pharmaceutical kit comprising the antibody of any one of claims 1 to 3, the immunoconjugate of any one of claims 9 to 14, or the pharmaceutical composition of claim 15.

Images