Her3 binding protein and use thereof

Abstract

An HER binding protein and a use thereof. An anti-HER3 antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the anti-HER3 antibody or the antigen-binding fragment thereof, and a method for preparing the anti-HER3 antibody or the antigen-binding fragment thereof. Compared to a positive control antibody BMK1, the anti-HER3 antibody or the antigen-binding fragment thereof has a stronger binding capability to a human HER3 protein and / or tumor cells expressing the human HER3 protein. A use of the antibody or the antigen-binding fragment thereof in treating and diagnosing diseases.

Description

HER3-binding proteins and their uses

[0001] Reference to relevant applications

[0002] This application claims priority to Chinese Patent Application No. 202510073488.9, filed on January 16, 2025, entitled “HER3 Binding Protein and Its Use Thereof,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of biomedicine. More specifically, this invention provides a HER3-binding protein, particularly a monoclonal antibody against HER3, especially a high-affinity chimeric antibody against HER3 and a humanized monoclonal antibody, and their applications.

[0004] Background of the Invention

[0005] Human epidermal growth factor receptor (HER) proteins are a family of receptor tyrosine kinases that function in both normal and tumor cells. This family consists of four highly homologous epidermal growth factor receptors (EGFR[ERBB1 / HER1]), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4), including a ligand-binding extracellular domain, a transmembrane domain, an intracellular kinase domain, and a C-terminal tail. Except for HER2, family members are usually activated by conformational changes induced by ligand binding, followed by homodimerization or heterodimerization among family members, ultimately leading to activation of the intracellular signaling cascade [1], resulting in increased cell viability and proliferation. HER3 is a unique member of the EGFR family with little or no intracellular tyrosine kinase activity, and its activation depends on heterodimerization with another receptor. Therefore, HER3 overexpression alone is not carcinogenic, but its expression is ubiquitous in various cancers, including lung cancer and other solid tumors [2], which makes it possible for targeted therapy in tumors. Moreover, some studies have pointed out that HER3 is a major determinant of resistance to some treatments targeting other ErbB receptors [3]. Given its ubiquitous expression in different solid tumors and its role in tumorigenesis and drug resistance, anti-HER3 monoclonal antibodies have great potential for development into drug forms such as ADCs, bispecific antibodies and CAR-T.

[0006] Currently, there are no HER3-targeting antibody drugs on the market. Therefore, it is urgent and necessary to develop HER3-targeting antibodies with higher specificity, lower toxicity, better clinical efficacy, and more convenient administration methods, which will provide patients with more treatment options. Summary of the Invention

[0007] In this application, the inventors developed chimeric and humanized antibodies targeting HER3 with superior properties. These antibodies specifically recognize / bind to HER3 and, compared to known positive control antibodies (BMK1), exhibit stronger binding to tumor cells expressing human HER3 (such as MDA-MB-453 cells) and / or the human target protein human ErbB3 / Her3 protein-His. This led to the following invention.

[0008] The antibody of the present invention

[0009] In another aspect, the present invention provides an isolated antibody that specifically binds to human HER3 or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and comprises a heavy chain CDR (HCDR1, HCDR2, and HCDR3) and a light chain CDR (LCDR1, LCDR2, and LCDR3) selected from any one of the following groups:

[0010] In some embodiments, at least one of the HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCR3 of the antibody contains a mutation, said mutation being a substitution, deletion, or addition of one or more amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids). In some embodiments, said substitution is a conserved substitution.

[0011] In some embodiments, the antibody is a polyclonal antibody. In some preferred embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0012] In some embodiments, the antibody comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region (VH) and the light chain variable region (VL) each comprise an amino acid sequence selected from any of the following groups or each comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher sequence identity with an amino acid sequence selected from any of the following groups:

[0013] In some implementations, the antibodies mentioned above are chimeric antibodies or humanized antibodies.

[0014] In some embodiments, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises a heavy chain constant region derived from or originating from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4), preferably, the heavy chain constant region is a heavy chain constant region derived from human IgG1, the amino acid sequence of which is shown in SEQ ID NO:388.

[0015] In some embodiments, the light chain of the antibody or antigen-binding fragment of the present invention comprises a light chain constant region derived from or originating from human immunoglobulins (e.g., κ or λ), preferably, the light chain constant region is a light chain constant region derived from human immunoglobulin κ, the amino acid sequence of which is shown in SEQ ID NO:389.

[0016] The antibody or antigen-binding fragment of the present invention binds to the K of the human HER3 antigen. D The value can be less than approximately 1 × 10 -7 M, preferably less than about 1×10 -8 M, more preferably less than about 1×10 -9 M, such as through full dynamic detection of surface plasmon resonance.

[0017] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments is selected from ScFv, Fab, Fab', (Fab')2, Fab'-SH, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multispecific antibody.

[0018] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments is labeled.

[0019] In some embodiments, the antibody or its antigen-binding fragment carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0020] Derived antibodies

[0021] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to HER3 (particularly human HER3). Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., an avidin or a multihistidine tag) capable of mediating the binding of the antibody or antigen-binding fragment to another molecule.

[0022] As a derivative of antibodies, the present invention provides a conjugate comprising the antibody or its antigen-binding fragment of the present invention and a conjugation portion.

[0023] In some embodiments, the coupling portion is selected from detectable markers. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads (e.g., The label may include thermometric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioisotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via connectors of varying lengths to reduce potential steric hindrance.

[0024] In some embodiments, the coupling portion is selected from therapeutic agents. In some embodiments, the therapeutic agent is preferably an antitumor agent, such as a cytotoxic agent, cytokine, toxin, or radionuclide.

[0025] In some embodiments, the described substance is a DNA topoisomerase I inhibitor, DX8951. In some embodiments, the coupling portion is linked to the antibody or its antigen-binding fragment via a linker, for example, the linker is MC-GGFG.

[0026] In some embodiments, the coupling portion is MC-GGFG-DX8951, which is formed by linking a DNA topoisomerase I inhibitor DX8951 and a protease-cleavable linker MC-GGFG, and has the following structural formula:

[0027] In some embodiments, the conjugation moiety is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups.

[0028] As a derivative of antibodies, the present invention provides a multispecific antibody comprising the antibody of the present invention or its antigen-binding fragment.

[0029] In some embodiments, the multispecific antibody comprises the antibody of the present invention or its antigen-binding fragment as a first antigen-binding domain, and further comprises at least one second antigen-binding domain targeting other targets.

[0030] In some embodiments, each antigen-binding domain of the multispecific antibody retains its original binding specificity.

[0031] In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0032] As a derivative of the antibody, the present invention provides a chimeric antigen receptor comprising the antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises the antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv) as an extracellular antigen-binding domain specifically binding to HER3, as well as a transmembrane domain and one or more intracellular T cell signaling domains. The present invention also provides host cells (e.g., immune cells such as T lymphocytes, NK cells) containing or expressing the chimeric antigen receptor.

[0033] Antibody preparation

[0034] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0035] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Additionally, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture media. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0036] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a variable region of the heavy chain and / or the variable region of the light chain of the present invention. According to codon degeneracy in the art, in some embodiments, the nucleotide sequence may be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0037] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, granule, bacteriophage, lentivirus, etc. In some embodiments, the vector is capable of expressing the antibody or antigen-binding fragment of the present invention in a subject (e.g., a mammal, such as a human).

[0038] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0039] In some embodiments, the antibody or antigen-binding fragment of the present invention can be used to construct a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain (e.g., ScFv) that specifically binds to HER3, a transmembrane domain, and one or more intracellular T-cell signaling domains. In such embodiments, the isolated nucleic acid molecule of the present invention may comprise a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further comprising a nucleotide sequence encoding an antibody or antigen-binding fragment (e.g., ScFv) of the present invention. In some embodiments, the isolated nucleic acid molecule of the present invention encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0040] In some embodiments, the antibodies or antigen-binding fragments of the present invention can be used to construct chimeric antigen receptor-modified immune cells, the chimeric antigen receptor-modified immune cells comprising chimeric antigen receptors (CARs) and immune cells (e.g., T lymphocytes, NK cells).

[0041] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell may be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., *E. coli*). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, Expi293F cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0042] In some embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may contain a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further containing a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv). In some embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor containing an antigen-binding fragment of an antibody of the present invention (e.g., ScFv).

[0043] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, comprising culturing the host cell of the present invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0044] Therapeutic applications

[0045] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule, a carrier, a host cell, a conjugate, a multispecific antibody, or a chimeric antigen receptor or a host cell expressing said chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient.

[0046] In some embodiments, the pharmaceutical compositions of the present invention comprise the antibody or antigen-binding fragment of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0047] In some embodiments, the pharmaceutical compositions of the present invention comprise the carrier or host cell of the present invention, and pharmaceutically acceptable carriers and / or excipients. In such embodiments, the isolated nucleic acid molecule comprised of the carrier comprises a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv); the host cell comprises the isolated nucleic acid molecule or carrier as described above. In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment of an antibody of the present invention (e.g., ScFv). In some embodiments, the host cell is an immune cell, such as a T cell. In some embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0048] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity. In some embodiments, the additional pharmaceutically active agent is selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0049] In some embodiments, the antibody or antigen-binding fragment of the present invention is provided as a separate component or as a mixed component with the additional pharmaceutically active agent. Therefore, the antibody or antigen-binding fragment of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0050] In some embodiments, the antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing said chimeric antigen receptor in the pharmaceutical composition of the present invention is sufficient (e.g., in a subject):

[0051] (1) Inhibit cell (e.g., tumor cells) proliferation;

[0052] (2) Inhibits tumor growth;

[0053] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0054] (4) Inhibit HER3-mediated signal transduction;

[0055] (5) Prevention and / or treatment of HER3-mediated diseases / conditions; or

[0056] (6) Any combination of (1)-(5) above.

[0057] In some embodiments, the HER3-mediated disease / condition is a tumor, such as a tumor expressing HER3. In some embodiments, the tumor is a solid tumor, such as lung cancer.

[0058] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing said chimeric antigen receptor, or pharmaceutical composition thereof in the preparation of a medicament for: inhibiting cell proliferation, or for the prevention and / or treatment and / or adjuvant treatment of tumors.

[0059] In some embodiments, the drug is used to inhibit the proliferation of cells expressing HER3 (e.g., tumor cells).

[0060] In another aspect, the present invention provides a method for inhibiting cell proliferation, comprising contacting the cells with an antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing the chimeric antigen receptor, or a pharmaceutical composition. In some embodiments, the cells are cells expressing HER3, such as tumor cells.

[0061] In another aspect, the present invention provides a method for preventing and / or treating and / or adjuvant treating tumors in a subject, the method comprising administering to a subject in need an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing said chimeric antigen receptor, or a pharmaceutical composition thereof.

[0062] In some embodiments, the method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In some embodiments, the second therapy may be applied simultaneously, separately, or sequentially with the methods described above.

[0063] In any of the above embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing said chimeric antigen receptor, or pharmaceutical composition of the present invention can be any tumor type. In some embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing said chimeric antigen receptor, or pharmaceutical composition of the present invention is a HER3-positive tumor. In some embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing said chimeric antigen receptor, or pharmaceutical composition of the present invention is a solid tumor, such as lung cancer.

[0064] The antibodies or antigen-binding fragments thereof of the present invention, and the pharmaceutical compositions of the present invention, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0065] Furthermore, the antibody or its antigen-binding fragment of the present invention may be present in the pharmaceutical composition in unit dose form for ease of administration.

[0066] The antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some preferred embodiments, the antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0067] The pharmaceutical compositions of the present invention may include an antibody or antigen-binding fragment thereof of the present invention in a "therapeutic effective amount" or a "preventive effective amount". A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0068] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0069] In this invention, the subject can be a mammal, such as a human.

[0070] Detection Application

[0071] The antibody or its antigen-binding fragment of the present invention can specifically bind to HER3, thereby enabling it to be used to detect the presence or level of HER3 in a sample.

[0072] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the antibody of the present invention or an antigen-binding fragment thereof is labeled with a detectable marker. In a partially preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable marker.

[0073] In this invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Particularly preferred is that such labels are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads (e.g., The method includes: 1) thermometric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads; and 2) biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, the detectable markers described above can be linked to the antibodies of the present invention via connectors of varying lengths to reduce potential steric hindrance.

[0074] In another aspect, the present invention provides a method for detecting the presence or level of HER3 in a sample, comprising the step of using an antibody or antigen-binding fragment of the present invention. In a partially preferred embodiment, the antibody or antigen-binding fragment of the present invention is further labeled with a detectable marker. In a partially preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment of the present invention using a reagent labeled with a detectable marker. The method may be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0075] In some embodiments, the method includes contacting the sample with the antibody or antigen-binding fragment of the present invention, and detecting the formation of the complex, under conditions that allow the antibody or its antigen-binding fragment to form a complex with HER3.

[0076] Given that HER3 is expressed at low levels or not at all in normal tissues, and expressed or expressed at high levels in some cancers, tumors can be diagnosed by detecting the presence or level of HER3 in a sample. Therefore, in some embodiments, the method is used to diagnose tumors, such as HER3-positive tumors, like lung cancer.

[0077] In some implementations, the method includes detecting the expression level of HER3 in a test sample from a subject and comparing the expression level to a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is an indication of tumor.

[0078] In another aspect, the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or level of HER3 in a sample and / or diagnosing tumors is provided.

[0079] In another aspect, the present invention provides diagnostic or therapeutic kits comprising the antibodies or antigen-binding fragments thereof described in this invention, nucleic acid molecules, vectors, host cells, conjugates, or multispecific antibodies, and instructions for use. The kits may also include a drug delivery device for local administration. The drug delivery device includes a drug-loaded syringe or a needleless device.

[0080] The antibodies of this invention exhibit high affinity and strong specificity for human HER3, inhibiting HER3-mediated signaling and cell proliferation. Therefore, the antibodies of this invention have the potential for tumor prevention and / or treatment. Furthermore, the antibodies of this invention can be chimeric or humanized antibodies, exhibiting stronger binding to human HER3 protein or tumor cells expressing HER3 protein compared to known positive control antibodies (BMK1). The chimeric antibody molecules of this invention mediate rapid internalization of HER3 target proteins on the surface of tumor cells, with a higher internalization rate than the positive control antibody (BMK1). Additionally, the conjugates of the chimeric antibodies and cytotoxins of this invention can mediate specific killing of tumor cells, showing comparable killing effects to BMK1 on most BT474 cells (see Example 4). The antitumor effect of the humanized antibody-cytotoxin conjugates (ADCs) of this invention is significantly better than or comparable to the positive control antibodies, demonstrating effective antitumor activity in vivo and possessing significant clinical value.

[0081] Brief description of the attached diagram

[0082] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0083] Figure 1 shows the FACS binding detection of the chimeric antibody to MDA-MB-453 cells (Figure 1A, 1B, 1C).

[0084] Figures 2A, 2B, and 2C show the ELISA results of the chimeric antibody binding to the human HER3 target protein.

[0085] Figures 3A, 3B, and 3C show the ELISA binding detection results of the chimeric antibody against monkey HER3 target protein.

[0086] Figures 4A, 4B, and 4C show the FACS endocytosis detection results of the chimeric antibody on MDA-MB-453 cells.

[0087] Figures 5A, 5B, and 5C show the FACS endocytosis detection results of the chimeric antibody on MCF-7 cells.

[0088] Figures 6A, 6B, 6C, and 6D show the in vitro killing activity of the chimeric antibody ADC molecule on BT474 cells.

[0089] Figure 7 shows the in vivo tumor-suppressing effects of humanized candidate molecules ADC 309-z6-p1-Dxd, 309-z6-p4-Dxd, 309-z8-p1-Dxd, 309-z16-p1-Dxd, 309-z17-p1-Dxd and control antibody BMK-Dxd in a mouse model of PC-9 human lung cancer subcutaneous transplantation tumors. The dosage was 3 mg / kg, and the frequency of administration was once every two weeks, for a total of 2 administrations.

[0090] Invention Details

[0091] definition

[0092] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0093] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0094] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0095] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, an antibody includes any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody therefore includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, camel antibodies, single-domain antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0096] As used herein, an “antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires immune-specific binding (i.e., exhibits at least or at least about 10 amino acids). 7 M -1 -10 8 M -1 Antibodies against the Ka antigen.

[0097] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0098] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those skilled in the art, a hybridoma can proliferate and continuously supply cells that produce a specific monoclonal antibody. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0099] As used herein, “conventional antibody” refers to an antibody containing two heavy chains (which may be labeled H and H') and two light chains (which may be labeled L and L') and two antigen-binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability (e.g., heavy chains include, but are not limited to, V). H Chain, V H -C H 1 chain and V H -C H 1-C H 2-C H 3 chains), and each light chain can be a full-length light chain or any functional area (e.g., light chains include, but are not limited to, V). L Chain and V L -C L Each heavy chain (H and H') is paired with a light chain (L and L', respectively).

[0100] As used in this article, full-length antibodies are antibodies with two full-length heavy chains (e.g., V). H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L Antibodies with the hinge region, such as antibodies naturally produced by B cells through antibody secretion and synthetically produced antibodies with the same structural domain.

[0101] As used in this article, dsFv refers to a stable V H -V L Fv of engineered intermolecular disulfide bonds.

[0102] As used herein, the Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure synthesized, for example, through recombinant methods. The Fab fragment contains a light chain (containing V...L and C L ) and another chain, the other chain containing a heavy chain variable region (V H ) and a constant region structural domain of the heavy chain (C H 1).

[0103] As used herein, the F(ab')2 fragment is an antibody fragment resulting from the digestion of immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure synthesized, for example, by a recombinant method. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain containing several additional amino acids, including cysteine ​​residues that form the disulfide bond connecting the two fragments.

[0104] As used in this article, the Fab' fragment is a fragment that contains half of the F(ab')2 fragment (one heavy chain and one light chain).

[0105] As used herein, scFv fragments refer to variable regions (V) of light chains covalently linked in any order via peptide linkers. L ) and heavy chain variable region (V H The antibody fragment has a linker length that allows the two variable domains to bridge with minimal interference. An exemplary linker is (Gly-Ser) with some Glu or Lys residues dispersed to increase solubility. n Residues.

[0106] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0107] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0108] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this invention also cover antibodies containing one or two amino acid mutations within the CDRs.

[0109] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0110] As used herein, a variable domain or variable region is a specific Ig domain of the antibody heavy or light chain, containing a variable amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region domain VL (also denoted as V). L ) and VH (or also represented as V) L Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), the CDR being part of the antigen-binding site domain.

[0111] As used herein, "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with a cognate antigen. Natural, conventional full-length antibody molecules have two conventional antigen-binding sites, each containing a variable region portion of the heavy chain and a variable region portion of the light chain. The conventional antigen-binding site contains a loop connecting antiparallel β-chains within the variable region domain. The antigen-binding site may contain other portions of the variable region domain. Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions are also called complementarity-determining regions (CDRs).

[0112] As used herein, “hypervariant region,” “HV,” “complementarity-determining region,” and “CDR” and “antibody CDR” are interchangeably used to refer to one of the multiple portions within each variable region that together form the antigen-binding site of the antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. For example, the light chain variable region domain contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3 (or LCDR1, LCDR2, and LCDR3); the heavy chain variable region domain contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3 (or HCDR1, HCDR2, and HCDR3). The three CDRs in the variable region are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loop of the parallel chain connecting the β-sheet of the variable region. Those skilled in the art know and can identify CDRs based on numbering methods such as Kabat or Chothia (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J.Mol.Biol. 196:901-917). In this invention, antibody CDRs are numbered using the Kabat method.

[0113] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0114] As used in this article, the "constant region" domain is a domain in the antibody heavy or light chain that contains a more conserved amino acid sequence than the variable region domain. In a typical full-length antibody molecule, each light chain has a single light chain constant region (C0). L ) structural domains, and each heavy chain contains one or more heavy chain constant regions (C H ) structural domain, including C H 1. C H 2. C H 3 and C H 4. Full-length IgA, IgD, and IgG isotypes include C. H 1. C H 2. C H 3 and hinge region, while IgE and IgM contain C H 1. C H 2. CH 3 and C H 4. C H 1 and C L The structural domain extends the Fab arm of the antibody molecule, thus facilitating interaction with antigens and the rotation of the antibody arm. The antibody constant region can serve effector functions, such as, but not limited to, clearing antigens, pathogens, and toxins specifically bound to the antibody, for example, through interactions with various cells, biomolecules, and tissues.

[0115] As used in this article, the functional region of an antibody is at least VH, VL, and C containing the antibody. H (e.g., C) H 1. C H 2 or C H 3) C L Or the hinge region structural domain or at least the antibody portion of its functional region.

[0116] As used herein, the functional region of the VH domain is at least a portion of the complete VH domain that retains at least a portion of the binding specificity of the complete VH domain (e.g., by retaining one or more CDRs of the complete VH domain), such that the functional region of the VH domain binds either alone or in combination with another antibody domain (e.g., V... L The VH domain (or a combination of its regions) binds to the antigen. An exemplary VH domain is a functional region containing CDR1, CDR2, and / or CDR3 of the VH domain.

[0117] As used herein, a functional region of a VL domain is at least a portion of an intact VL domain that retains at least a portion of the binding specificity of the intact VL domain (e.g., by retaining one or more CDRs of the intact VL domain), such that the functional region of the VL domain binds the antigen alone or in combination with another antibody domain (e.g., a VH domain) or a region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2, and / or CDR3 of the VL domain.

[0118] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶. 7 M -1 Or 1x 10 8 M -1 Or a larger affinity constant Ka (or 1 x 10⁻⁶) -7 M or 1×10 -8M or a lower dissociation constant (K) d The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0119] As used herein, the term "competition" with respect to antibodies refers to a first antibody or its antigen-binding fragment binding to an epitope in a manner sufficiently similar to that of a second antibody or its antigen-binding fragment, such that the binding of the first antibody to its associated epitope is detectably reduced in the presence of the second antibody compared to the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody; this is possible but not necessary. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, in cases where each antibody detectably inhibits the binding of another antibody to its associated epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes. Both competing and cross-competing antibodies are covered in this invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or fragment thereof), those skilled in the art will recognize, based on the teachings provided in this invention, that such competing and / or cross-competing antibodies are covered in this invention and can be used in the methods disclosed herein.

[0120] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms "polypeptide" and "protein" are used interchangeably herein.

[0121] "Isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is not associated with its natural associated component in its native state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur naturally. Therefore, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system of a different natural source cell will be "isolated" from its natural associated component. Isolation can also render a protein substantially free of its natural associated component, i.e., using protein purification techniques well known in the art.

[0122] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0123] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are typically linked together by phosphodiester bonds.

[0124] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0125] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0126] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of that nucleic acid.

[0127] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0128] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0129] “Codon optimization” refers to methods of modifying nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence). Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be customized to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See Nakamura. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0130] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0131] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0132] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0133] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sundørland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0134] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0135] As used herein, the term “prevention” refers to methods implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a tumor) in a subject.

[0136] As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonging survival compared to the expected survival (if no treatment was received).

[0137] As used herein, the terms "subject" or "object" refer to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor, or is at risk of having the aforementioned disease.

[0138] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0139] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0140] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0141] As used herein, the term "effector function" refers to the biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant), which vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, and the half-life / clearance of antibodies and antigen-antibody complexes. Methods for altering antibody effector functions are known in the art, for example, by introducing mutations into the Fc region.

[0142] As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (such as natural killer (NK) cells, neutrophils, or macrophages), enabling these cytotoxic effector cells to specifically bind to target cells to which the antigen is attached, and then kill the target cells by secreting cytotoxins.

[0143] As used herein, the term "antibody-mediated internalization" refers to the phenomenon of an antibody crossing the cell membrane after binding to a cell surface antigen. Internalization includes antibody-mediated receptor (e.g., HER3) internalization.

[0144] In this document, combination therapy includes the use of an anti-HER3 antibody or an antigen-binding fragment thereof covered by the present invention in combination with one or more other active therapeutic agents of a second therapy (e.g., chemotherapeutic agents) or other preventive or therapeutic modalities (e.g., radiotherapy).

[0145] In such combination therapies, the various active agents often have different complementary mechanisms of action, and the combination therapy may lead to a synergistic effect. Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. Combination therapies may allow for a reduction in the dosage of one or more agents in the regimen to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or preventative effect on underlying diseases, conditions, or symptoms.

[0146] In this document, "combination" includes therapies that can be administered separately, such as those formulated separately for individual administration (e.g., those provided in kits), and therapies that can be administered together as a single formulation (i.e., "co-formulation"). In some embodiments, the anti-HER3 antibody or its antigen-binding fragment of the present invention may be administered sequentially. In other embodiments, the anti-HER3 antibody or its antigen-binding fragment may be administered simultaneously. The antibody or its antigen-binding fragment of the present invention may be used in combination with at least one other (active) agent in any manner.

[0147] In this study, HER3 positivity was determined by immunohistochemistry and staining intensity evaluation performed by a professional clinical pathologist.

[0148] The terms "cancer" and "tumor" are used interchangeably to refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0149] As used herein, when the terms “about” or “approximately” are used with a numerical variable, they generally mean that the value of the variable is within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% or more of the specified value. Detailed Implementation

[0150] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0151] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0152] Example 1. Generation of antibody hybridomas

[0153] 1.1 Material Preparation

[0154] 1) Preparation of plasmid for DNA immunization: The full-length DNA sequence encoding ERBB3 (HER3) (NP_001973.2) (SEQ ID NO:396) was synthesized at Genewiz (Suzhou, China) and then subcloned into the p13 (proprietary) vector. After confirming the sequence accuracy, 10 mg of plasmid (concentration >2 mg / mL, endotoxin <30 EU / mg) was prepared. After sequencing verification, the plasmid was used for DNA immunization.

[0155] 2) Preparation of control antibody: The amino acid sequence encoding the variable region of the positive control antibody (BMK1, patritumab, sequence derived from patent CN118767159A, SEQ ID.3 / SEQ ID.4) was synthesized at Genewiz (Suzhou, China). The light and heavy chains were then subcloned into pcDNA3.4 expression vectors containing the constant regions of human kappa and IgG1 subtypes, respectively. Plasmids containing the antibody heavy chain variable region (VH) and light chain variable region (VL) genes were co-transfected into Expi293F cells using Lipofectamine™ 2000 transfection reagent (Invitrogen, 11668019). After culturing for 5 days, the supernatant was collected. The sample was purified using an AKTA sample introduction system and a protein A (MabSelect SuRe, GE-17543804) affinity chromatography column to obtain the purified antibody.

[0156] 3) Human ErbB3 / Her3 Protein-Fc (ER3-H5259) for immunotherapy was purchased from Bipsys.

[0157] 1.2 Animal immunization and serum titer detection

[0158] All mice were housed in a barrier system. Four 6-8 week old SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used as the first group, and four 6-8 week old BALB / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were used as the second group. In the first group of four rats, the antigen was Human ErbB3 / Her3Protein-Fc, and the immunization dose was 10 μg / rat, administered via paw puncture and subcutaneous injection every week. In the second group of four mice, the antigen was a DNA immunization plasmid, and the immunization dose was 100 μg / mouse, administered via intramuscular and intradermal injection every week. The adjuvant mixture included alum adjuvant (Pierce, 77161), CpG-ODN (self-made), and Titer-Max immunizing adjuvant (Sigma, H0262). Blood was collected from the animals after every two injections, and serum titers against the target protein HER3 were measured by ELISA.

[0159] ELISA assay: First, a 384-well flat-bottom microplate was coated with rabbit anti-His-tagged (C-terminus) antibody and incubated overnight at 4°C. After blocking at 25°C for 1 hour, 0.0625 μg / mL of Human ErbB3 / Her3 Protein-His (ACRO ER3-H5223) was added to the plate and incubated at 25°C for 1 hour. After washing, animal serum at different dilutions was added to the plate (starting with a 1:100 dilution, followed by a 3-fold serial dilution), and incubated at 25°C for 2 hours. The plate was washed, and then HRP-labeled goat anti-mouse IgG-Fc detection antibody was added and incubated at 25°C for 1 hour. After washing 6 times, TMB substrate was added, and after 5 minutes of color development, the reaction was terminated by adding 1M H2SO4. The absorbance was read at 450 nm using a microplate reader. Experimental data were analyzed using GraphPad Prism 7 software.

[0160] 1.3 Cell Fusion

[0161] After seven immunizations, all animals exhibited strong serum titers of the target antigen. Two mice from each group with the highest serum titers were given a booster immunization with 20 μg / mouse of protein antigen. Three days (72 hours) later, the animals were euthanized, and lymph node and spleen cells were collected for plasma B cell sorting and cell fusion. Sp2 / 0 myeloma cells (ATCC, CRL-1581) in the logarithmic growth phase were collected. TMThe cells were mixed with plasma B cells in electrofusion buffer and then electrofused according to a standard electrofusion procedure. The fused cells were resuspended in DMEM medium containing a mixture of 20% FBS, 1×HAT (hypoxanthine), aminopterin, and thymidine (Sigma, H0262), and 1×OPI (Sigma, O5003), and cultured at 37°C in a 5% CO2 incubator for 10 days.

[0162] Example 2. Hybridoma Screening

[0163] Ten days after fusion, the culture supernatant from 96-well plates was used to detect the binding of Human ErbB3 / Her3 Protein-His antigen using enzyme-linked immunosorbent assay (ELISA). Based on the fusion titer results, positive hybridoma cells binding to Human ErbB3 / Her3 Protein were selected for a second round of screening. Flow cytometry fluorescence sorting (FACS) was used to detect the binding and internalization activities of the hybridoma supernatant with MDA-MB-453 cells (ATCC, HTB-131). Simultaneously, ELISA was used to detect the binding activity of the hybridoma supernatant with monkey Recombinant Rhesus HER3 / ERBB3 Protein (His Tag) (SinoBiological, catalog number: 90043-K08H) and mouse ErbB3 / Her3 Protein (ACROBiosytems Group, catalog number: ER3-M52H5) antigen proteins.

[0164] Based on the results of the second round of screening, 20 positive hybridoma cell lines were selected for semi-solid subclonal culture. The binding activity of the supernatant of hybridoma monoclonal cells with Mouse ErbB3 / Her3 Protein (ACRO ER3-M52H5) was detected by enzyme-linked immunosorbent assay (ELISA), and its binding activity with MDA-MB-453 cells was detected by flow cytometry.

[0165] The ELISA screening method is the same as in 1.2. The FACS and internalization experimental procedures are as follows:

[0166] 1) FACS testing:

[0167] MDA-MB-453 tumor cells were incubated with hybridoma supernatant at 4°C for 1 hour. After washing the cells, Alex647 fluorescein-labeled goat anti-mouse IgG-Fc detection antibody was added, and the cells were incubated at 4°C in the dark for 30 minutes. The median fluorescence intensity (Median FI) of the cells was measured using flow cytometry, and the experimental data were analyzed using GraphPad Prism 7 software.

[0168] 2) Internalization assay (FACS):

[0169] Flow cytometry fluorescence sorting (FACS) was used to detect hybridoma supernatant-mediated internalization of cell surface targets. MDA-MB-453 and MCF-7 tumor cells (ATCC, HTB22) were mixed with hybridoma supernatant and incubated for 1 hour at 4°C. Human IgG1 isotype antibody was used as a negative control. After washing the cells, Alexa647 fluorescently labeled goat anti-human detection antibody was added. Cells were incubated at 4°C in the dark for 30 minutes. After washing the cells, they were resuspended in culture medium and divided into two aliquots, which were incubated at 37°C and 4°C, respectively, for 4 hours in 5% CO2. After washing, the cells were resuspended in acid wash solution and incubated at 4°C for 6 minutes. After washing and resuspending the cells, the median fluorescence intensity (Median FI) of the cells was detected and analyzed by flow cytometry. Experimental data were calculated using a four-parameter nonlinear fitting method in GraphPad Prism 7 software to calculate EC50. 50 value.

[0170] Example 3. Hybridoma sequencing

[0171] Based on ELISA and FACS results, 20 positive monoclonal hybridoma cells meeting the requirements were selected for expanded culture. Total RNA was extracted from the hybridoma cells according to standard methods. After reverse transcription into cDNA samples using a reverse transcription kit, the cDNA of the heavy chain variable region and light chain variable region of the antibody was amplified by PCR using hybridoma sequencing primers. After purification and recovery, the PCR product fragments were subcloned into T vectors. Clones were selected for sequencing. Some sequences were repetitive, and finally, 19 pairs of chimeric antibody molecular sequences were obtained.

[0172] Example 4. Generation and characterization of chimeric antibodies

[0173] 4.1 Generation of chimeric antibodies

[0174] Sequencing yielded the antibody heavy chain variable region (VH) and light chain variable region (VL) sequences. The frame region sequences are shown in Table 1. The CDR region sequences in Table 1 are defined using the Kabat system. Amino acid sequences derived from the antibody variable regions obtained from hybridoma sequencing were synthesized at Genewiz (Suzhou, China). The light and heavy chains were then cloned into pcDNA3.4 (purchased from Invitrogen, A14697) expression vectors containing the constant regions of human kappa and IgG1 subtypes, respectively. The constructed light and heavy chain expression plasmids were transformed into E. coli DH5α competent cells. Single colonies were picked, and after successful sequencing, plasmids were extracted in large quantities to obtain chimeric antibody light chain and chimeric antibody heavy chain expression plasmids.

[0175] Plasmids containing antibody heavy chain variable region (VH) and light chain variable region (VL) genes and Lipofectamine were used. TM Expi293F cells were co-transfected with 2000 transfection reagent (Invitrogen, 11668019) and cultured for 5 days. The supernatant was then collected. The sample was purified using an AKTA sample introduction system and a protein A affinity chromatography column (MabSelect SuRe, GE-17543804) to obtain purified antibodies.

[0176] 4.2 Characterization of chimeric antibodies

[0177] 4.2.1 Detection of chimeric antibody binding (FACS):

[0178] Flow cytometry fluorescence sorting (FACS) was used to detect the binding of chimeric antibodies to human ErbB3 / Her3 Protein-His. MDA-MB-453 tumor cells were incubated at 4°C for 1 hour with different concentrations of monoclonal antibodies (starting from 100 nM and serially diluted 3.16-fold to 0.0003 nM). BMK1 was used as a positive control, and human IgG1 isotype antibody was used as a negative control. After washing the cells, Alexa647 fluorescently labeled goat anti-human detection antibody was added, and the cells were incubated at 4°C in the dark for 30 minutes. The median fluorescence intensity (Median FI) of the cells was detected and analyzed using flow cytometry. The experimental data were calculated using a four-parameter nonlinear fitting method in GraphPad Prism 7 software to calculate EC50. 50 value.

[0179] Figure 1 shows the binding results of 19 chimeric antibodies on MDA-MB-453 tumor cells. The results indicate that all chimeric antibodies have strong binding to MDA-MB-453, and the binding is higher than that of the positive control antibody (BMK1).

[0180] 4.2.2 Detection of human / cynomolgus monkey binding of chimeric antibodies

[0181] The binding of chimeric antibodies to human target protein Human ErbB3 / Her3 Protein-His and monkey target protein Recombinant Rhesus HER3 / ERBB3 Protein (His Tag) was determined by enzyme-linked immunosorbent assay (ELISA). 384-well clear flat-bottom ELISA plates were pre-coated with rabbit anti-his tag (C-terminus) antibody and incubated overnight at 4°C. After blocking at 25°C for 1 hour, 0.0625 μg / mL of antigen Human ErbB3 / Her3 Protein-His or Recombinant Rhesus HER3 / ERBB3 Protein (His Tag) was added to the plates, and incubation was carried out at 25°C for 1.5 hours. After washing, different concentrations of chimeric monoclonal antibody (serially diluted 3.16-fold from 30 nM to 0.0001) were added, and incubation was carried out at 25°C for 1.5 hours. BMK1 was used as a positive control, and human IgG1 isotype antibody as a negative control. After washing, HRP-labeled goat anti-human IgG detection antibody was added, and the mixture was incubated at 25°C for 1 hour. After washing, TMB was added for color development, and the reaction was terminated by adding 1M H2SO4. The absorbance was read at 450 nm using a microplate reader. Experimental data were calculated using a four-parameter nonlinear fitting method in GraphPad Prism 7 software to determine the EC50. 50 value.

[0182] Figure 2 shows the binding results of 19 chimeric antibodies to the human target protein HER3. All candidate chimeric antibodies showed strong binding to the human target protein Human ErbB3 / Her3 Protein-His, and the binding was significantly higher than that of the control antibody (BMK1).

[0183] Figure 3 shows the binding results of the candidate chimeric antibodies to the monkey target protein Recombinant Rhesus HER3 / ERBB3. All candidate chimeric antibodies showed strong binding, demonstrating that all candidate chimeric antibodies possess human-monkey cross-linking properties.

[0184] 4.2.3 Detection of internalized chimeric antibodies (FACS)

[0185] Flow cytometry fluorescence sorting (FACS) was used to detect the internalization of target proteins on the surface of MDA-MB-453 and MCF-7 cells mediated by monoclonal antibodies. The specific method is described in Example 2.

[0186] As shown in Figure 4, the candidate chimeric antibody molecules were able to mediate the rapid internalization of the HER3 target protein on the surface of tumor cells MDA-MB-453. All antibodies exhibited superior endocytosis compared to the control antibody (BMK1), with antibodies 1.28.6-xIgG1K, 1.33.9-xIgG1K, 1.69.2-xIgG1K, 1.267.15-xIgG1K, 1.309.1-xIgG1K, 1.324.14-xIgG1K, and 1.331.7-xIgG1K demonstrating better HER3 internalization.

[0187] Figure 5 shows the results of chimeric antibody internalization in MCF-7 cells. The candidate chimeric antibody molecules were able to mediate the rapid internalization of HER3 target protein on the surface of MCF-7 tumor cells. Most of the chimeric antibodies mediated strong MCF-7 internalization, such as 1.28.6-xIgG1K, 1.33.9-xIgG1K, 1.69.2-xIgG1K, 1.267.15-xIgG1K, 1.309.1-xIgG1K, 1.324.14-xIgG1K, and 1.331.7-xIgG1K. The internalization activity of all antibodies was higher than that of the control antibody (BMK1).

[0188] 4.2.4 In vitro killing assay of chimeric antibodies

[0189] The preferred chimeric antibody was conjugated to MC-GGFG-DX8951 (MCE, HY-114233) via random cysteine ​​conjugation, with a DAR value of approximately 8, for cell killing detection. The structure of MC-GGFG-DX8951 is as follows:

[0190] BT474 (ATCC, HTB-20) cells were spaced at 6 × 10⁶ cells per well. 3 The sample was seeded into 96-well cell culture plates, 80 μL per well, and cultured overnight. The next day, 20 μL of different concentrations of ADC samples (starting at a working concentration of 1000 nM, serially diluted 4-fold to 0.0610 nM) were added to each well, and the plates were cultured for 6 days. After culture, 50 μL of CellTiter-Glo (Promega, G7570) was added to each well, and the plates were immediately shaken for 5 minutes to mix thoroughly. The fluorescence intensity (RLU) of each well was then read using an Envision multi-function plate reader. Antibody-dependent cytotoxicity was calculated using the following formula: Cytotoxicity % = 100 * (RLU cell only - RLU sample) / RLU cell only. The data were analyzed using a four-parameter nonlinear fitting method to calculate EC50 using GraphPad Prism 7 software. 50The results are shown in Figure 6. Of the 17 selected candidate chimeric antibodies, except for 1.6.11 and 1.298.10, all showed comparable killing effects to BMK1 on BT474 cells, EC50. 50 The concentration ranged from 289 to 477 nM, with a maximum kill rate of 47% to 92%. The ADC (hIgG1-Isotype-Dxd) used as an isotype control only showed some non-specific kill at high concentrations.

[0191] Example 5. Humanized Antibody

[0192] 5.1 Humanization design, removal of post-translational modification sites, and plasmid construction

[0193] Based on the performance of the chimeric antibody, the 1.309.1 chimeric antibody was selected for humanization. The original murine antibody sequence was compared with fully human antibody sequences in the IMGT database to identify a human germline sequence with the highest homology in the framework region, which was used as the template for humanization. The murine antibody CDR region sequence was grafted into the selected humanized template framework to obtain the humanized antibody 1.309.1-z0. Key sites in the framework region that might maintain the CDR conformation were selected for reversion mutations, and these sites were combined to obtain multiple humanized variants. The presence of post-translational modification (PTM) sites in the 1.309.1 sequence was investigated. Several PTM-removed variants were constructed. These humanized and PTM-removed variants were used to synthesize antibody plasmids for recombinant expression via Genewiz (Suzhou, China): the heavy chain variable region sequence was synthesized and constructed into a plasmid containing human IgG heavy chain CH1, hinge, CH2, and CH3 fragments; the light chain variable region sequence was synthesized and constructed into a plasmid containing human IgK light chain constant region fragments. The constructed plasmids had the same constant region but contained different site mutations in the heavy and light chain variable regions. The humanized antibody heavy chain variable region sequence (VH) and light chain variable region sequence (VL), and the frame region sequence are shown in Table 2 attached.

[0194] 5.2 Production of humanized antibodies

[0195] Plasmids containing the antibody heavy chain variable region (VH) and light chain variable region (VL) genes were co-transfected into Expi293F cells. After 6 days of cell culture, the supernatant was collected. Using the AKTA sample loading system, the supernatant was automatically loaded onto a protein A affinity chromatography column (MabSelect SuRe, GE-17543804). After loading, the column was washed with 0.1M Tris-HCl, pH 7.0 equilibration buffer, followed by elution and collection of the target protein with 0.1M glycine, pH 3.5 elution buffer. Protein purity was analyzed by SDS-PAGE and HPLC-SEC, both showing a purity greater than 90%.

[0196] Example 6. Characterization of humanized antibodies

[0197] 6.1 SPR detection of humanized antibodies

[0198] The affinity of humanized antibody molecules for binding to Human ErbB3 / Her3 Protein-His was detected using SPR technology with Biacore 8K.

[0199] Humanized antibody molecules were captured by anti-human IgG Fc antibodies coated on a CM5 chip. Human ErbB3 / Her3 Protein-His (0, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 nm) diluted in seven consecutive serial numbers and buffer were injected through the chip channels at a rate of 30 μL / min. The binding time was 180 s, and the dissociation time was 600 s. All data were fitted using a 1:1 binding model, and the results are shown in Table 3. Except for 309-z6-p4, the affinity of other humanized candidate molecules for the HER3 antigen was close to that of chimeric antibodies.

[0200] Table 3. Affinity of antibody to human HER3 antigen as determined by surface plasmon resonance kinetics.

[0201] 6.2 Results of humanized antibody-cell affinity assay (FACS)

[0202] Five humanized antibody molecules and chimeric antibody precursors were analyzed by flow cytometry. MDA-MB-453 cells were cultured at a density of 5 x 10⁻⁶ cells / mL. 4Cells were transferred at a density of 100 μL / well to 96-well U-shaped plates. 100 μL of a 2-fold serially diluted 400 μg / mL initial antibody sample was added to each well, and the plates were incubated at 4°C for 1 hour. Then, 100 μL of FITC-conjugated Affini Pure Goat Anti-Human IgG, FcγFragment Specific fluorescent secondary antibody (Jackson ImmunoResearch Inc., 109-095-008) was added to each well, and the plates were incubated at 4°C for 0.5 hours in the dark. After incubation, the supernatant was removed, and the cells were resuspended in 180 mL of 1X PBS + 1% BSA in each well. Fluorescence signals were detected using a BD Canto II flow cytometer.

[0203] A standard curve was generated using the Quantum™ FITC-5MESF Kit (Bangs laboratories-555) to quantify the number of fluorescent molecules. A linear regression standard curve was fitted using Graphpad Prism 7. Fluorescence signals in the samples were detected by flow cytometry (BD Canto II), and the data were analyzed using FlowJo. The number of bound and free IgG molecules in the samples was calculated based on the standard curve. K was calculated using the Scatchard plot method combined with Graphpad Prism 7. D The results are shown in Table 4. The results show that all humanized antibody molecules have considerable cell-binding ability with the chimeric antibody parent.

[0204] Table 4. Results of FACS binding detection of purified antibody

[0205] 6.3 In vivo efficacy studies of humanized candidate ADC molecules

[0206] MC-GGFG-DX8951 (Dxd, purchased from MCE, HY114233), a compound formed by linking the DNA topoisomerase I inhibitor DX8951 and the protease-cleavable linker MC-GGFG, was chemically conjugated to humanized candidate antibody molecules and control antibodies to prepare 309-z6-p1-Dxd, 309-z6-p4-Dxd, 309-z8-p1-Dxd, 309-z16-p1-Dxd, 309-z17p1-Dxd, and BMK-Dxd, with a DAR value of approximately 4. A human lung cancer Pc-9 cell line subcutaneous transplantation tumor model was established by subcutaneously inoculating Pc-9 cells into the right back of Balb / c nude mice, with an average tumor size of 98 mm. 3Mice were randomly divided into 7 groups, including a load cell control group: 5 mice in each group were administered the drug once every two weeks for a total of two administrations. Tumor size was measured twice a week. The results are shown in Figure 7.

[0207] The tumor-suppressing effects of 309-z6-p4-Dxd and 309-z8-p1-Dxd were significantly better than those of the positive control antibody, while the tumor-suppressing effect of 309-z6-p1-Dxd was comparable to that of the positive control antibody, indicating that the humanized candidate molecules have effective anti-tumor activity in vivo.

[0208] The antibody sequence information involved in this article is as follows:

[0209] Reference body: BMK1

[0210] >BMK1-HC (The underlined sequence is the constant region of the antibody heavy chain)

[0211] >BMK1-LC (The underlined sequence is the constant region of the antibody light chain)

[0212] The light and heavy chain sequences of the control antibody BMK1 are referenced to SEQ ID NO:3 and SEQ ID NO:4 in patent application CN118767159A.

[0213] In the embodiments of the present invention, the antibody variable region sequence information is shown in Tables 1 and 2 below. The amino acid sequences of the heavy chain constant region and light chain constant region of the antibody are identical to the amino acid sequences of the heavy chain constant region and light chain constant region of the control antibody BMK1, respectively. That is, the heavy chain constant region sequences of the chimeric antibody and the humanized antibody are:

[0214] The light chain constant region sequences of chimeric antibodies and humanized antibodies are as follows:

[0215] Receptor tyrosine protein kinase erbB-3 isotype 1 precursor [human]

[0216] >P21860(NP_001973.2)

[0217] Table 5. Serial numbers corresponding to the six CDRs of the antibody of this invention

[0218] References

[0219] [1]Sheng Q, Liu J. The therapeutic potential of targeting the EGFR family in epithelial ovarian cancer. Br J Cancer 2011;104(8):1241–5doi 10.1038 / bjc.2011.62.[PubMed:21364581]

[0220] [2]Ocana A, Vera-Badillo F, Seruga B, Templeton A, Pandiella A, Amir E. HER3 overexpression and survival in solid tumors: a meta-analysis. J Natl Cancer Inst 2013;105(4):266–73doi 10.1093 / jnci / djs501.[PubMed:23221996]

[0221] [3] Amin DN, Campbell MR, Moasser MM. The role of HER3, the unpretentious member of the HER family, in cancer biology and cancertherapeutics. Semin Cell Dev Biol. 2010; 21(9):944–50.

Claims

1. An antibody that specifically binds to HER3 or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and comprises a heavy chain CDR and a light chain CDR selected from any one of the following groups: Or, at least one of the HCDR1, HCDR2, HCR3, LCDR1, LCDR2, LCR3 of the antibody contains a mutation, said mutation being a substitution, deletion, or addition of one or more amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody, preferably a chimeric antibody, and more preferably a humanized antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the substitution is a conserved substitution.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region (VH) and the light chain variable region (VL) respectively comprise an amino acid sequence selected from any of the following groups or respectively comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with an amino acid sequence selected from any of the following groups:

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, for example, the sequence of the heavy chain constant region is as shown in SEQ ID NO:388 and / or the sequence of the light chain constant region of the antibody is as shown in SEQ ID NO:

389.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', (Fab')2, Fab'-SH, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is labeled, such as a detectable label, for example an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof binds to the K group of the human HER3 antigen. D The value is less than approximately 1 × 10 -7 M, preferably less than about 1×10 -8 M, more preferably less than about 1×10 -9 M, such as through full dynamic detection of surface plasmon resonance.

9. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8.

10. A vector comprising the nucleic acid molecule of claim 9; preferably, the vector is a cloning vector or an expression vector.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-8, comprising culturing the host cell of claim 11 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

13. A conjugate comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-8 and a conjugate portion thereto; preferably, the conjugate portion is selected from detectable markers (such as radioactive isotopes, fluorescent substances, luminescent substances, colored substances or enzymes) or therapeutic agents (such as cytotoxic agents, cytokines, toxins or radionuclides).

14. The conjugate of claim 13, wherein the conjugation portion is a DNA topoisomerase I inhibitor DX8951.

15. The conjugate of claim 13 or 14, wherein the conjugation portion is connected to the antibody or its antigen-binding fragment via a connector, for example, the connector being MC-GGFG.

16. The conjugate of claim 13, wherein the conjugated portion is MC-GGFG-DX8951, formed by linking DX8951 and the protease-cleavable linker MC-GGFG, having the following structural formula:

17. A multispecific antibody comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8; Preferably, the multispecific antibody comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-8 as a first antigen-binding domain, and further comprises at least one second antigen-binding domain targeting other targets; Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

18. A chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof (e.g., ScFv) as described in any one of claims 1-8, a transmembrane domain, and one or more intracellular T cell signaling domains.

19. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-8, or an isolated nucleic acid molecule according to claim 9, or a carrier according to claim 10, or a host cell according to claim 11, or a conjugate according to any one of claims 13-16, or a multispecific antibody according to claim 17, or a chimeric antigen receptor or a host cell expressing the chimeric antigen receptor according to claim 18, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity; Preferably, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs, or any combination thereof; Preferably, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the other pharmaceutically active agent.

20. A diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, or an isolated nucleic acid molecule as claimed in claim 9, or a vector as claimed in claim 10, or a host cell as claimed in claim 11, or a conjugate as claimed in any one of claims 13-16, or a multispecific antibody as claimed in claim 17, or a chimeric antigen receptor or a host cell expressing said chimeric antigen receptor as claimed in claim 18, or a pharmaceutical composition as claimed in claim 19, and optionally instructions for use and / or a delivery device.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the isolated nucleic acid molecule according to claim 9, or the vector according to claim 10, or the host cell according to claim 11, or the conjugate according to any one of claims 13-16, or the multispecific antibody according to claim 17, or the chimeric antigen receptor or host cell expressing the chimeric antigen receptor according to claim 18, or the pharmaceutical composition according to claim 19 in the preparation of a medicament for inhibiting cell (e.g., cells expressing HER3, such as tumor cells) proliferation or for the prevention and / or treatment and / or adjuvant treatment of tumors; Preferably, the antibody or its antigen-binding fragment, isolated nucleic acid molecule, carrier, host cell, conjugate, multispecific antibody, or pharmaceutical composition is administered in combination with other pharmaceutically active agents, for example, simultaneously, separately, or sequentially. Preferably, the additional pharmaceutically active agent is a drug with antitumor activity; Preferably, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

22. The use according to claim 21, wherein the tumor is a HER3-positive tumor; Preferably, the tumor is a solid tumor, such as lung cancer.

23. A method for inhibiting cell proliferation, comprising contacting the cells with an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, or an isolated nucleic acid molecule as claimed in claim 9, or a vector as claimed in claim 10, or a host cell as claimed in claim 11, or a conjugate as claimed in any one of claims 13-16, or a multispecific antibody as claimed in claim 17, or a chimeric antigen receptor as claimed in claim 18 or a host cell expressing the chimeric antigen receptor, or a pharmaceutical composition as claimed in claim 19; Preferably, the cells are cells that express HER3, such as tumor cells, like lung cancer cells.

24. A method for preventing and / or treating and / or adjuvant treating tumors in a subject, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, or an isolated nucleic acid molecule as claimed in claim 9, or a vector as claimed in claim 10, or a host cell as claimed in claim 11, or a conjugate as claimed in any one of claims 13-16, or a multispecific antibody as claimed in claim 17, or a chimeric antigen receptor or a host cell expressing said chimeric antigen receptor as claimed in claim 18, or a pharmaceutical composition as claimed in claim 19.

25. The method of claim 24, further comprising administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; Optionally, the second therapy may be applied simultaneously, separately, or sequentially with the method of claim 24.

26. The method of claim 24 or 25, wherein, The tumor is a HER3-positive tumor; Preferably, the tumor is a solid tumor, such as lung cancer.

27. A method for detecting the presence or level of HER3 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment of any one of claims 1-8 under conditions that allow the formation of a complex between the antibody or its antigen-binding fragment and HER3, and detecting the formation of the complex; Preferably, the method is used to diagnose tumors, such as HER3-positive tumors, such as solid tumors like lung cancer; Preferably, the method includes detecting the expression level of HER3 in a test sample from a subject and comparing the expression level with a reference value, wherein an increase in the expression level compared with the reference value is an indicator of tumor.

28. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the isolated nucleic acid molecule according to claim 9, or the vector according to claim 10, or the host cell according to claim 11, or the conjugate according to any one of claims 13-16, or the multispecific antibody according to claim 17 in the preparation of a detection kit, said kit being used to detect the presence or level of HER3 in a sample and / or to diagnose tumors; Preferably, the tumor is a HER3-positive tumor; Preferably, the tumor is a solid tumor, such as lung cancer.

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