Anti-HER3 antibody, antibody-drug conjugate containing same, and uses thereof

Abstract

An anti-HER3 antibody, or an antibody-drug conjugate (ADC) containing an anti-HER3 antibody, is provided. Also provided is the use of the antibody or ADC in the treatment of HER3-expressing cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to PCT International Application PCT / CN2021 / 099998, filed June 15, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to anti-HER3 antibodies. The present disclosure also relates to antibody-drug conjugates containing the anti-HER3 antibodies and their use in the treatment of HER3-expressing cancers. [Background technology]

[0003] HER3 is a member of the ERBB family and plays a key role in cell proliferation, tumor metastasis, and drug resistance. Although drugs targeting EGFR and HER2 have shown great clinical efficacy in mitigating many cancers, previous efforts to develop anti-HER3 antibodies for cancer therapy have repeatedly failed, suggesting that addressing HER3 and its associated pathways alone may not be sufficient to inhibit tumor growth. Consistent with this hypothesis, U3-1402, an ADC targeting HER3, has shown promising results in early-phase clinical trials in breast and NSCLC. Furthermore, bispecific antibodies manipulating both HER3 and HER2 significantly reduce disease biomarkers in some enriched populations. The latest clinical advances suggest that HER3 remains a promising tumor target, provided there are additional mechanisms. Summary of the Invention

[0004] In one aspect, the present invention provides an anti-HER3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

[0005] In some embodiments, the VH comprises a CDR-H1 of SEQ ID NO: 15, a CDR-H2 of SEQ ID NO: 17, and a CDR-H3 of SEQ ID NO: 19.

[0006] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.

[0007] In some embodiments, the VL comprises a CDR-L1 of SEQ ID NO:5, a CDR-L2 of SEQ ID NO:7, and a CDR-L3 of SEQ ID NO:9.

[0008] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 12, with or without the leader sequence of SEQ ID NO: 13, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0009] In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:2, with or without the leader sequence of SEQ ID NO:3, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0010] In some embodiments, the anti-HER3 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0011] In some embodiments, the heavy chain of the anti-HER3 antibody is of the IgG1 type.

[0012] In some embodiments, the anti-HER3 antibody is a humanized antibody and the heavy chain comprises the amino acid sequence of SEQ ID NO: 21, 25 or 27, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0013] In some embodiments, the anti-HER3 antibody is a humanized antibody and the light chain comprises the amino acid sequence of SEQ ID NO:23, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0014] In some embodiments, the HER3 is human or monkey HER3.

[0015] In some embodiments, the anti-HER3 antibody inhibits NRG1-induced phosphorylation of HER3.

[0016] In some embodiments, the anti-HER3 antibody has an EC 50 It binds to human or monkey HER3.

[0017] In some embodiments, the anti-HER3 antibody has internalizing activity upon binding to HER3.

[0018] In another aspect, the present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the above VH and / or the above VL.

[0019] In some embodiments, the isolated nucleic acid is selected from SEQ ID NOs: 1, 11, 22, 24, 26, and 28.

[0020] In another aspect, the disclosure provides a vector comprising the isolated nucleic acid.

[0021] In another aspect, the disclosure provides a host cell comprising the isolated nucleic acid or vector.

[0022] In another aspect, the disclosure provides a host cell expressing an anti-HER3 antibody, or an antigen-binding fragment thereof.

[0023] In another aspect, the disclosure provides an antibody conjugate comprising an anti-HER3 antibody, or antigen-binding fragment thereof, linked to a chemical moiety.

[0024] In some embodiments, the anti-HER3 antibody, or antigen-binding fragment thereof, is attached to the chemical moiety via a linker.

[0025] In some embodiments, the linker is enzymatically cleavable.

[0026] In some embodiments, the linker comprises a Val-Cit moiety.

[0027] In some embodiments, the antibody conjugate is an antibody drug conjugate (ADC).

[0028] In some embodiments, the chemical moiety is a radioisotope, a chemotherapeutic agent, or a cytotoxic drug.

[0029] In some embodiments, the cytotoxic agent is a toxin.

[0030] In some embodiments, the toxin is selected from auristatin E, auristatin F, MMAE, and MMAF.

[0031] In another aspect, the disclosure provides a pharmaceutical composition comprising an anti-HER3 antibody, or antigen-binding fragment thereof, or antibody conjugate, and a pharma- ceutically acceptable carrier.

[0032] In some embodiments, the pharmaceutical composition further comprises one or more other anti-cancer agents.

[0033] In another aspect, the disclosure provides the use of an anti-HER3 antibody, or antigen-binding fragment thereof, or antibody conjugate in the manufacture of a medicament for the treatment of cancer.

[0034] In some embodiments, the cancer expresses HER3.

[0035] In some embodiments, the cancer is gastric cancer or colon cancer.

[0036] In another aspect, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-HER3 antibody or antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition.

[0037] In some embodiments, the cancer expresses HER3.

[0038] In some embodiments, the cancer is gastric cancer or colon cancer. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows that murine 3F8 specifically binds to SP2 / 0-HER3 cells. [Diagram 2] FIG. 1 shows the binding affinity of mouse 3F8 to human HER3, HER2, and EGFR as measured by ELISA. [Diagram 3] FIG. 1 shows that murine 3F8 recognizes both human and monkey HER3 with similar potency as measured by ELISA. [Figure 4] FIG. 1 shows that murine 3F8 inhibits NRG1-induced phosphorylated HER3. [Diagram 5] FIG. 1 shows that murine 3F8 is rapidly taken up by cells with varying surface levels of HER3. [Figure 6] FIG. 1 shows that anti-HER3 antibodies efficiently inhibit tumor growth in a BT474 subcutaneous xenograft model. [Figure 7] FIG. 1 shows [89Zr]Zr-ch3F8 imaging of gastric PDX model GAS078. [Figure 8]FIG. 1 shows representative [89Zr]Zr-ch3F8 imaging in six PDX models. [Figure 9] FIG. 1 shows that ch3F8-MMAE maintains similar binding affinity to ch3F8. [Figure 10] FIG. 1 shows the cytotoxicity of ch3F8-MMAE in multiple cell lines. [Figure 11] FIG. 1 shows that ch3F8-MMAE inhibits tumor growth in the gastric model GAS078. [Figure 12A] Figure 1 shows that hu3F8 maintains binding affinity after heat, acid and repeated freeze-thaw stress tests. Three clones of hu3F8 were incubated at pH 3.5 for 0, 2, 4 and 6 hours and then proceeded to ELISA assay to measure binding affinity. [Figure 12B] Figure 1 shows that hu3F8 maintains binding affinity after heat, acid and repeated freeze-thaw stress tests. Three clones of hu3F8 were incubated at 40°C for various days and then proceeded to ELISA assay to measure binding affinity. [Figure 12C] Figure 1 shows that hu3F8 maintains binding affinity after heat, acid and repeated freeze-thaw stress testing. Three clones of hu3F8 were freeze-thawed for 3 or 5 cycles and then carried on to ELISA assays to measure binding affinity. [Figure 13A] FIG. 1 shows that hu3F8-MMAE inhibited tumor growth in a dose-dependent manner. [Figure 13B] FIG. 1 shows that hu3F8-MMAE has little effect on body weight. [Figure 14] FIG. 1 shows that hu3F8-MMAE inhibits tumor growth in the gastric PDX model GAS078 at 10 mg / kg with a single injection. [Figure 15] FIG. 1 shows that hu3F8-MMAE inhibits tumor growth in gastric PDX model GAS078 at 6 mg / kg. [Figure 16]FIG. 1 shows that hu3F8-MMAE inhibits tumor growth in the colon PDX model CS226. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Any methods, devices and materials similar or equivalent to those described herein can be used to practice the present invention. The following definitions are provided to facilitate understanding of certain terms used herein and are not intended to limit the scope of the present disclosure.

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0042] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, without regard to the presence or absence of the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0043] Human epidermal growth factor receptor 3 (HER3), also known as receptor tyrosine protein kinase erbB-3 (ERBB3), is a member of the EGFR / ERBB family. Unlike other ERBB family members HER2 and EGFR, HER3 itself does not possess kinase activity. Therefore, HER3 must associate as a heterodimer with either of its kinase-active members, EGFR or HER2, to trigger downstream activities. Upon binding to its natural ligand NRG1, HER3 undergoes conformational changes, heterodimerization, and phosphorylation, signaling to activate MAPK, PI3K / Akt, and PLCγ. At the same time, HER3 also exerts its biological activity in a ligand-independent manner in the presence of high levels of HER2. HER3 plays an important role in cell growth and proliferation, embryonic development, and tumorigenesis. HER3 knockout mice are severely growth-deprived and are lethal at embryonic day 13.5. HER3 also contributes to drug resistance to drugs targeting a variety of proteins and indications.

[0044] The term "antibody" generally refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment thereof (antigen-binding fragment) that retains the essential epitope-binding characteristics of an Ig molecule. In a full-length antibody, each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region generally consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, the CDRs of the heavy chain are referred to as CDR-H1, CDR-H2, and CDR-H3, respectively, from the amino-terminus of the heavy chain, while the CDRs of the light chain are referred to as CDR-L1, CDR-L2, and CDR-L3, respectively, from the amino-terminus of the light chain. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In a broad sense, the term "antibody" also refers to scFvs or sdAbs that are not derived from immunoglobulin molecules having four polypeptide chains. The term "antibody" further refers to any multispecific antibody (particularly a bispecific antibody) comprising an anti-HER3 antibody or an antigen-binding fragment thereof.

[0045] An antibody or a functional fragment of an antibody may have one or more modified amino acid residues, for example, the heavy or light chain of the antibody has one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid, and deletion of one or two amino acids from the carboxyl group.

[0046] An "antigen-binding fragment" is a portion of an antibody, such as, for example, F(ab')2, Fab, Fv, scFv, sdAb, etc. Antigen-binding fragments of a full-length antibody retain the target specificity of the full-length antibody. Thus, recombinant functional antibody fragments, such as single chain variable chain fragments (scFvs), have been used to develop therapeutics as an alternative to mAb-based therapeutics. The scFv fragment (approximately 25 kDa) consists of two variable domains, namely VH and VL. Naturally, the VH and VL domains tend to non-covalently associate and dissociate through hydrophobic interactions. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create scFvs.

[0047] As used herein, the term "single domain antibody" (sdAb) has its general meaning in the art and refers to a single heavy chain variable domain of an antibody of the type found in camelid mammals, which naturally lacks light chains. Such single domain antibodies are HSingle domain antibodies are also called "H" or "nanobodies". The amino acid sequence and structure of single domain antibodies can be considered to be composed of four framework regions (FR1, FR2, FR3, and FR4) and three complementarity determining regions (CDR1, CDR2, and CDR3). Single domain antibodies can therefore be defined as amino acid sequences with the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which are similar to the variable domains VH or VL. Compared to using conventional antibodies or other antibody fragments (e.g., scFv), the use of sdAbs as single antigen-binding proteins or as antigen-binding domains in larger proteins or polypeptides offers a number of important advantages. The advantages of sdAbs include that only a single domain is needed to bind antigen with high affinity and high selectivity, sdAbs are highly stable to denaturants or conditions including heat, pH, and proteases, and sdAbs can access targets and epitopes that are inaccessible to conventional antibodies. Typically, sdAbs are produced in camelids such as llamas, but can also be produced synthetically using techniques well known in the art.

[0048] The term "chimeric antibody" refers to an antibody that comprises a variable region, i.e., a binding region, of murine origin and at least a portion of a constant region derived from a different source or species (e.g., human), and is usually prepared by recombinant DNA techniques. Chimeric antibodies that comprise a murine variable region and a human constant region are particularly preferred. Such murine / human chimeric antibodies are usually the product of expressed immunoglobulin genes that comprise a DNA segment encoding a murine immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques now well known in the art.

[0049] The term "humanized antibody" refers to an antibody whose framework or "complementarity determining regions" (CDRs) have been modified to include CDRs of an immunoglobulin with a different specificity compared to that of the parent immunoglobulin. In a preferred embodiment, the VH and VL CDRs are grafted into the framework regions of a human antibody to prepare a "humanized antibody". The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be those of naturally occurring human antibodies. If necessary, the framework regions can be modified by further mutations. Particularly preferred CDRs correspond to the CDRs that represent sequences recognizing the antigens mentioned above for the chimeric antibodies. Preferably, such humanized versions are chimerized with human constant regions. As used herein, the term "humanized antibody" also includes such antibodies that have been modified in the constant region, for example by "class switching", i.e. by changes or mutations in the Fc part (e.g. from IgG1 to IgG4 and / or IgG1 / IgG4 mutations), to generate properties according to the invention, in particular with regard to C1q binding and / or FcR binding.

[0050] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies or selected human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germline immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. Human antibodies can also be produced in phage display libraries.

[0051] As used herein, the term "anti-HER3 antibody" refers to an antibody that specifically binds to human HER3 antigen. An antibody that "specifically binds" to an antigen of interest, i.e., HER3, is an antibody that can bind to the antigen with sufficient affinity so that the antibody is useful for targeting cells expressing the antigen. Binding affinity can be measured using standard binding assays such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden).

[0052] The term "sequence identity" with respect to a peptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference peptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0053] The term "internalization", when used in relation to the binding of the antibody of the present invention to the HER3 antigen on the surface of a cancer cell, refers to the rapid uptake of the antibody-antigen complex from the external environment by receptor-mediated endocytosis, micropinocytosis, phagocytosis or other similar cellular uptake and / or transport pathways. Thus, in one embodiment, the "internalization" of the antibody of the present invention relates to the uptake of the antibody from the external environment by mechanisms including plasma membrane folding and vesicle formation. When the antibody of the present invention is conjugated to a chemical moiety such as a radioisotope, a fluorophore or a cytotoxin, the chemical moiety can be internalized into HER3-expressing cells together with the antibody of the present invention. Whether or not an antibody has internalization activity can be confirmed by a method generally known to those skilled in the art, for example, by contacting a label-binding anti-HER3 antibody with a HER3-expressing cell and confirming whether the label (e.g., a radioisotope, a fluorophore, or a fluorescent protein) is incorporated into the cell, or by contacting a cytotoxic substance-binding anti-HER3 antibody with a HER3-expressing cell and confirming whether the death of the HER3-expressing cell is induced. More specifically, the internalization activity of an anti-HER3 antibody can be measured, for example, by the method described in the Examples. An anti-HER3 antibody having an internalization activity can be used as a pharmaceutical composition such as an anticancer agent described later, for example, by binding to a cytotoxic substance.

[0054] As used herein, the term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0, or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell, but also the progeny of such cells. Such progeny may not be identical to the parent cell, since certain modifications may occur in successive generations due to mutations or environmental influences, but are still included within the scope of the term "host cell."

[0055] As used herein, the term "nucleic acid" refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-natural analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-natural analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the bonds between them include non-natural synthetic analogs, such as, but not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences (i.e., A, U, G, C) in which "U" replaces "T".

[0056] As used herein, the term "isolated nucleic acid" refers to a purified state, and in such context means that the nucleic acid is substantially free of other biological molecules such as proteins, lipids, carbohydrates, or other substances such as cellular debris and growth medium.

[0057] "EC 50 The term "half maximal effective concentration," also known as the half maximal effective concentration, refers to the concentration of an antibody or antigen-binding portion thereof that gives a half-maximal response in an assay, for example, by FACS or ELISA.

[0058] As used herein, the term "antibody conjugate" refers to an antibody or antigen-binding fragment thereof conjugated to another chemical moiety, such as a radioisotope, a chemotherapeutic agent, and a toxin. In some embodiments, the chemical moiety is an isotope or a fluorophore, and thus the conjugated antibody can be used to reveal cells, tissues, or organs (including tumors) that express HER3 through in vivo imaging. In some embodiments, the antibody conjugate is an antibody drug conjugate (ADC).

[0059] The terms "anti-HER3 antibody drug conjugate" and "anti-HER3 ADC", used interchangeably herein, refer to an antibody drug conjugate comprising an antibody that specifically binds to HER3 and is linked to a cytotoxic drug (e.g., auristatin) via a linker. In general, an antibody (e.g., an anti-HER3 antibody) can retain biological activity, such as binding affinity to its target antigen, after being modified to become an ADC molecule. The term "cytotoxic drug" refers to a substance that inhibits or prevents the expression activity, function, and / or causes destruction of a cell. This term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof). Chemotherapeutic agents are well known in the art and include, but are not limited to, anthracyclines (e.g., daunorubicin (daunomycin, rubidomycin), doxorubicin, epirubicin, idarubicin, and valrubicin), anthracenediones (anthraquinones) such as mitoxantrone and pixantrone; platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin); tamoxifen and its metabolites, such as 4-hydroxytamoxifen (afimoxifen) and N-desmethyl-4-hydroxytamoxifen (endoxifen); paclitaxel (taxol) ), taxanes such as docetaxel, cabazitaxel, 10-deacetylbaccatin; alkylating agents (e.g., nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil); ethylenimines and methylmelamines (e.g., alkylsulfonates such as hexamethylmelamine, thiotepa, busulfan, nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U), and streptozoein (streptozotocin), and triazenes such as decarbazine (DTIC, dimethyltriazenoimidazole carboxamide);Antimetabolites (e.g., folic acid analogs such as methotrexate (amethopterin), pyrimidine analogs such as fluorouracil (5-fluorouracil, 5-FU), floxuridine (fluorodeoxyuridine, FUdR), and cytarabine (cytosine arabinoside), and purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, 6-TG), and pentostatin (2'-deoxycohonysine); natural products (e.g., vinblastine (VLB) and vin Vinca alkaloids such as cristine, epipodophyllotoxins such as etoposide and teniposide, and antibiotics such as dactinomycin (actinomycin D), bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin Q); enzymes such as L-asparaginase; biological response modifiers such as interferon alpha; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenal cortical suppressants such as mitotane and aminoglutethimide;Examples of cytotoxic drugs include auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoids, ricin, ricin A chain, combrestatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin, Examples of anti-HER3 antibody-drug conjugates include, but are not limited to, exotoxin (PE)A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogenin, restrictocin, phenomycin, enomycin, chrysin, crotin, calicheamicin, Sapaonaria officinalis inhibitors, glucocorticoids and other chemotherapeutic agents, and radioisotopes. As used herein, the term "auristatin" refers to a family of antimitotic agents. Auristatin derivatives are also included within the definition of the term "auristatin". Examples of auristatins include, but are not limited to, auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of dolastatins. In one embodiment, the anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-MMAE (e.g., ch3F8-MMAE or hu3F8-MMAE). In some embodiments, the linker is attached to a Cys residue in the hinge region of the antibody;

[0060] The term "pharmaceutical composition" refers to a formulation that is in a form so as to effect the biological activity of the active ingredients and thus can be administered to a subject for therapeutic use.

[0061] The term "pharmaceutical acceptable carrier" refers to any inert substance suitable for use in a formulation for delivering an active ingredient, such as an antibody or ADC of the present invention. The carrier may be a binder, coating, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial agent, or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutical acceptable carriers include water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol), dextrose, vegetable oil (such as olive oil), saline, buffer solution, buffered saline, and isotonic agents (such as sugars, polyalcohols, sorbitol, and sodium chloride).

[0062] The term "effective amount" or "therapeutically effective amount" refers to an amount of an active agent sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., which can be readily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the dosing regimen to be followed, whether it is administered in combination with other therapeutic agents, the timing of administration, the tissue to be imaged, and the physical delivery system that carries it.

[0063] As used herein, the phrase "other anti-cancer agents" refers to anti-cancer agents other than the anti-HER3 antibodies or anti-HER3 ADCs disclosed herein. Non-limiting examples of other anti-cancer agents include chemotherapeutic agents such as 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, doxorubicin, etoposide, carboplatin, cisplatin, cyclophosphamide, melphalan, dacarbazine, taxol, camptothecin, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin, and combinations thereof; biotherapeutic agents such as antibodies against PD-L1, PD-1, CTLA-4, CCR4, OX40; ionizing radiation; and cellular therapeutic agents such as chimeric antigen receptor (CAR) modified T cells or NK cells.

[0064] The terms "cancer" and "tumor", as used interchangeably herein, include, for example, lung cancer, non small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, central nervous system cancer, "CNS" refers to neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Preferably, such cancers are breast cancer, lung cancer, head and neck cancer, or pancreatic cancer, preferably lung cancer, head and neck cancer, or pancreatic cancer. Preferably, such cancers are further characterized by HER3 expression or overexpression, more preferably HER3 overexpression.

[0065] As used herein, the term "treat" or "treatment" means the treatment or management of a disease or condition in a subject, such as a mammal (particularly a human), including: (a) preventing the onset of a disease or condition, such as prophylactic treatment of a subject; (b) ameliorating a disease or condition, such as eliminating or resolving a disease or condition in a subject; (c) inhibiting a disease or condition, for example, by slowing or halting the progression of the disease or condition in a subject; or (d) alleviating the symptoms of a disease or condition in a subject.

[0066] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals, and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein.

[0067] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary, and the scope of the present invention will be limited only by the appended claims, and the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0068] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within that stated range, is encompassed within the scope of the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0069] Materials and Methods: Materials: The following reagents were purchased from Southern Biotech and used at the indicated dilutions: goat anti-mouse IgG-HRP (1030-05, 1:5000 dilution), goat anti-human IgG-PE (2040-09, 1:1000 dilution), goat anti-human kappa IgG-HRP (2061-05, 1:20000 dilution), goat anti-rabbit IgG-HRP (4030-05, 1:5000 dilution), streptavidin-FITC (7100-02, 1:500 dilution), mouse anti-human kappa-APC (9230-11, 1:500 dilution), mouse IgG-APC (0107-11, 0.1 mg / ml). NRG1 was obtained from Origene (TP723155). Cell culture media Roswell Park Memorial Institute (RPMI) 1640, Dulbecco's modified eagle's medium (DMEM), and fetal bovine serum were obtained from Hyclone. Recombinant patritumab was prepared in-house. HER3 antibody 3F8 was produced in-house from hybridoma or recombinant. m3F8 denotes mouse 3F8, ch3F8 and hu3F8 denote chimeric 3F8 and humanized 3F8, respectively.

[0070] Cell culture: SP2 / 0, SP2 / 0-HER3, SP2 / 0-HER2, SP2 / 0-EGFR, NCI-N87, MDA-MB-468, MDA-MB-453, 7901, HT29, MCF-7, and SK-BR-3 cells used in the study were purchased from ATCC and maintained in appropriate media as recommended by ATCC. SP2 / 0-EGFR, SP2 / 0-HER2, and SP2 / 0-HER3, which stably express human EGFR, HER2, and HER3, respectively, were generated in-house.

[0071] Generation of anti-HER3 hybridomas: On day 1, BALB / c mice (female, 8-10 weeks old) were inoculated with 1-2 x 10 SP2 / 0-HER3 cells expressing human HER3. 6Each mouse was intraperitoneally injected with 100 μg of cells in Freund's complete adjuvant. On the 8th day, a booster immunization was performed with the same amount of cells in Freund's incomplete adjuvant. From the 14th day, the mouse was immunized with the above amount of cells every 3 days, which was repeated three times. Three days after the last immunization, B lymphocytes were isolated from the spleen and fused with immortalized myeloma cells NS-1 cells to generate hybridoma cells.

[0072] Hybridoma cells were cultured in serial dilutions in 96-well plates, and the supernatants were collected and screened for antibodies that recognize HER3 expressed on the surface of SP2 / 0 cells by flow cytometry or recombinant HER3 by ELISA.

[0073] DNA cloning and sequencing of antibody variable regions: Briefly, total RNA extracted from hybridomas was reverse transcribed into first strand cDNA using Trizol (ThermoFisher). Then, 5' Rapid Amplification of Complementary DNA (5'RACE) followed by nested PCR was applied to amplify DNA sequences encoding variable regions, as described in the instructions of 5'RACE kit (Invitrogen, 18374-058). PCR products were cloned into pGM-T vector. Positive clones were proceeded to DNA sequencing, from which the corresponding protein sequences were deduced. Amino acids of variable regions were analyzed with Kabat numbering scheme.

[0074] Antibody expression: Briefly, DNA encoding the antibody heavy and light chains was cloned into the expression vector pCDNA3.1(+) (Invitrogen) and expressed in 293T cells. Antibodies were purified on Protein A or G columns (GE).

[0075] Humanization: Humanization was performed with GenScript. First, a mouse-human chimeric antibody (ch3F8) was generated by replacing the constant region of the heavy chain of the mouse antibody with the sequence of human IgG1 constant region and the constant region of the light chain of the mouse antibody with the sequence of human Igκ constant region. Then, humanization process was performed on the chimeric antibody according to the procedure in reference (Kuramochi et al). Residues in the mouse framework that are essential for maintaining affinity and specificity were preserved in generating the humanized antibody by replacing the mouse framework with human germline framework.

[0076] Codon optimized DNA sequences encoding the humanized antibodies were synthesized using GenScript.

[0077] Antibody expression and purification: ExpiCHO-S cells (cat. no. A29133, Gibco) transfected with plasmids carrying the coding DNA sequences of the indicated antibodies were grown and maintained in ExpiCHO medium (cat. no. A2910001, Gibco) at 32°C and 5% CO2 for 12 days. After spinning at 4000g for 30 min, the supernatant was collected and filtered through a 0.22 μm membrane. Protein A-bound antibodies (cat. no. 17508001, GE) were washed with 20 mM sodium phosphate (pH 7.0) and eluted with 0.1 M glycine (pH 3.0) as detailed in the manufacturer's manual. The eluted fractions were neutralized with 0.1 M Tris buffer (pH 9.0) and then switched to PBS buffer by ultrafiltration centrifugation. Protein concentration was measured by BCA.

[0078] Surface plasmon resonance (SPR): Kinetics and affinity were measured on a Biacore T200. Briefly, recombinant human HER3 antibody was immobilized on a Protein A chip (GE, Cat. No. 29-1275-55). Antigen was passed over the chip at concentrations ranging from 50 nM to a final concentration of 0.78125 nM, generated by two-fold serial dilution, and affinity and kinetics were measured.

[0079] FACS: Cultured cells were digested with 0.25% trypsin-EDTA and then spun at 1500 rpm for 5 min. The cell pellet was diluted with 5x10 PBS containing 5% FBS and 0.2% ProClin 300 in FACS solution. 6 The pellet was reconstituted with 100 μL of FACS solution containing goat anti-mouse IgG-PE (1:1000 dilution) and incubated on ice for 1 hour in the dark. The cells were then washed twice and resuspended in 200 μL of FACS solution. 50 μL of the cell suspension was incubated with 100 μL of primary antibody at a concentration of 1 μg / ml for 1 hour on ice. The pellet was reconstituted with 100 μL of FACS solution containing goat anti-mouse IgG-PE (1:1000 dilution) and incubated on ice for 1 hour in the dark. The cells were then washed twice and resuspended in 200 μL of FACS solution.

[0080] Western Blotting: For Western blotting, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes. Primary antibodies were: anti-HER2 (Cell Signaling, Catalog No.: 2165S), anti-HER3 (Cell Signaling, Catalog No.: 12708), anti-p-HER3 (Cell Signaling, Catalog No.: 4791), anti-β-actin (Cell Signaling, Catalog No.: 4967).

[0081] ELISA: Human HER2-ex-huFc, human HER3-huFc, and human EGFR-his were diluted to 2 μg / mL, 50 μL / well in a 96-well plate and incubated overnight at 4 °C. Washed with 0.5 × PBST, incubated with 100 μL of blocking buffer (PBS + 3% BSA) for 2 h at 37 °C, and washed with 0.5 × PBST. 3F8, serially diluted 1:3 in blocking buffer, was added at 50 μL / well and incubated at 37 °C for 40-50 min, then washed with 0.5 × PBST. Goat anti-mouse IgG-HRP (SouthernBiotech, 1030-05) was diluted 1:20000 in blocking buffer, 50 μL / well, incubated in the dark for 30 min, then washed with 0.5 × PBST. 50 μL of 1:1 mixed luminol buffer A+B was added to each well before detection.

[0082] NRG1-induced HER3 phosphorylation: Cells were cultured in 6-well plates and used for experiments when they reached 80% confluence. On the day of the experiment, cells were washed twice with PBS and incubated in serum-free medium for 6 hours, followed by treatment with 10 μg / mL of antibody overnight. To induce HER3 phosphorylation, NRG1 was added to a working concentration of 100 ng / mL, and cells were harvested for Western blot after 30 minutes.

[0083] Cytotoxicity: The day before the experiment, cells were seeded at 5000 / well in 96-well plates. 3F8-MMAE was added to the cells in a 1:3 dilution to working concentrations ranging from 100 nM to 1 pM, resulting in triplicate samples per single concentration. After 72 hours, cytotoxicity was measured using the ATPLite kit as described in the manufacturer's instructions.

[0084] Antibody stress test: 5mg / mL purified antibodies were stored at 4℃ as usual, incubated at 40℃ for 7 and 14 days for thermal stability evaluation, regenerated and kept in glycine solution at pH 3.5 for 2, 4 and 6 hours for acid stability evaluation, or subjected to 4 or 6 cycles of freeze-thaw treatment for freeze-thaw stability evaluation. Aggregation was measured by SEC-HPLC and binding affinity was measured by ELISA.

[0085] [ 89 Zr]Zr-antibody labeling: DFO-NCS was conjugated to the antibody and label as described in reference (Zeglis and Lewis, 2015). Briefly, DFO and antibody were mixed in a 5:1 molar ratio and incubated at 37 °C for 1 h. DFO-conjugated antibody was purified by SEC-HPLC.

[0086] 89 0.8 mCi of Zr-oxalate solution was mixed with DFO-conjugated antibody (0.2 mg / ml) in HEPES / Na2CO2 buffer (pH 7.0-7.5) and incubated at room temperature for 30 min. Radiochemical purity was assessed by TLC. Rf was [ 89 Zr]Zr-antibody 0-0.3, free 89The Rf of Zr is 0.6 to 1.0.

[0087] PET imaging: Approximately 100uCi 89 [Zr]Zr-antibody was administered intravenously to each animal, and images were collected and analyzed using a small animal PET imaging device at the indicated times after injection.

[0088] Preparation of 3F8-vc-MMAE and U3-1402: ADC preparation was performed by a CRO. Briefly, disulfide bonds in the antibody were reduced using TCEP (tris-2-carboxyethyl-phosphine) and DTPA (diethylenetriaminepentaacetic acid) reagents at 25°C for 1-3 hours. MC-VC-MMAE or MC-GGFG-Dxd were added dropwise to the reduced antibody solution and incubated at 25°C for 1-4 hours with gentle stirring. The DAR (drug-antibody ratio) of 3F8-vc-MMAE was about 3.8, whereas the DAR of U3-1402 (patritumab-GGFG-Dxd) was about 8.0. Both drugs were conjugated to cysteine ​​residues in the antibody.

[0089] The final product was purified by ultrafiltration. The purity and DAR were assessed by SEC-HPLC and HIC-HPLC, respectively.

[0090] In vivo efficacy study: Animals were maintained and used in accordance with IACUC guidelines. BALC / b nude and NPG mice, purchased separately from Charles River and SPF Biotech, were housed at 25°C with a 12-h dark / light cycle and free access to food and drink. Patient derived xenograft (PDX) models were generated by subcutaneous implantation of cryopreserved tissue fragments. Tumor sizes between 100 and 200 mm were included. 3 Drug treatment was started at the time when the 1000 mm 3 Animals were euthanized at a size of 0.05 mm. Gross health was observed daily. Tumor size and body weight were monitored and recorded every 3 days.

[0091] Software: Data were analyzed with Olinda, GraphPad Prism 6.0 or EXCEL. Example 1

[0092] Mouse 3F8 were incubated with SP2 / 0 wild type cells, or cells overexpressing HER3, HER2, or EGFR. Binding intensity was detected using a PE anti-mouse secondary antibody on a FACS machine.

[0093] The results are shown in Figure 1. 3F8 specifically binds to SP2 / 0-HER3 cells, but does not bind to other cells. Example 2

[0094] The binding affinity of mouse 3F8 to human HER3, HER2, and EGFR was measured by ELISA.

[0095] The results are shown in Figure 2. Mouse 3F8 recognizes only HER3, but does not recognize HER2 or EGFR. Example 3

[0096] The EC50 of murine 3F8 binding to human HER3, HER2, and EGFR was analyzed using GraphPad Prism 6.0. The results are shown in Table 1. 3F8 showed strong binding affinity in the subnanomolar range. [Table 1] Example 4

[0097] The species selectivity of murine 3F8 for human, monkey, rat and mouse HER3 was determined by ELISA.

[0098] The results are shown in Figure 3. 3F8 recognized both human and monkey HER3 with similar potency, but did not recognize mouse HER3. Example 5

[0099] EC of mouse 3F8 binding to human, monkey, rat, and mouse HER3 50was analyzed using GraphPad Prism 6.0. The results are shown in Table 2. 3F8 showed comparable binding affinity to human and monkey HER3 in the subnanomolar range. [Table 2] Example 6

[0100] Murine 3F8 inhibited NRG1-induced phosphorylated HER3. NCI-N87, MDA-MB-468, and MDA-MB-453 were treated with the HER3 ligand NRG1 to induce downstream phosphorylation of HER3. The effect of murine 3F8 in inhibiting NRG1-induced p-HER3 was measured by Western blotting. 3D4, an anti-HER3 antibody previously shown to compete for the binding of NRG1 to HER3, was applied as a positive control.

[0101] The results are shown in Figure 4. The data showed that 3F8 reduced phosphorylated HER3 protein levels but had no effect on total HER3 protein levels. Western blot of HER2 also showed that 3F8 had no effect on HER2 protein levels. Example 7

[0102] Mouse 3F8 is rapidly taken up by cells with varying surface levels of HER3. Cells with varying surface levels of HER3 were incubated with mouse 3F8 for 1 or 4 hours on ice as a control or at 37° C. The internalization rate was determined by subtracting the cell surface signal of the 37° C. incubation from the ice incubation control.

[0103] The results are shown in Figure 5. The data showed that 3F8 was rapidly internalized into cells, with the majority being internalized within 1 h of incubation, and the amount of the intracellular fraction increasing slightly when the incubation time was extended to 4 h, suggesting that 3F8 endocytosis is a rapid and continuous process. Example 8

[0104] Anti-HER3 antibodies efficiently inhibited tumor growth in a BT474 subcutaneous xenograft model. Anti-HER3 antibodies m3F8, m3D4, or a combination of m3F8+m3D4 were administered intravenously at 25 mg / kg every other week for 3 weeks. Tumor size was monitored every 3-4 days.

[0105] The results are shown in Figure 6. Both m3F8 and m3D4 significantly inhibit tumor growth (one-way ANOVA, p<0.05), but 3F8 shows better efficacy. The combination of m3F8 and m3D4 is as efficient as m3F8 alone. Both 3F8 and 3D4 are anti-HER3 antibodies. m3F8 represents a murine antibody. Example 9

[0106] [ 89 The gastric PDX model GAS078 was imaged using Zr]Zr-ch3F8. 89 [Zr]Zr-ch3F8 was administered intravenously to the gastric model GAS078. Images were collected at 4, 24, 48, 72, 96, and 168 hours after injection. Radiouptake in all organs was analyzed by Olinda and expressed as %ID / g (percentage of injected dose per gram of tissue).

[0107] Figure 7 shows the [ 89 Representative imaging of [Zr]Zr-ch3F8. Ch3F8 indicates chimeric 3F8 antibody. Data show the [ 89 We demonstrated a gradual increase in the uptake of [Zr]Zr-ch3F8. Tumor uptake remained stable up to 96 h post-injection, then decreased slightly at 168 h post-injection.

[0108] The data are also shown in Table 3. The [ 89 The uptake of Zr]Zr-ch3F8 was assessed as %ID / g (% injected dose / gram tissue). [Table 3] Example 10

[0109] [ 89 Multiple PDX models were imaged using Zr-ch3F8. 89 Zr]Zr-ch3F8 was injected intravenously. Images were collected 72 hours after injection. 89 Representative images of [Zr]Zr-ch3F8 imaging are shown in Figure 8. HER3 expression levels in tumor tissues as measured by ELISA and radiation uptake in major organs and tumor tissues 72 hours after injection are shown in Table 4. Significant tumor uptake was observed in all six PDX models tested.

[0110] Table 4. HER3 expression levels in tumor tissues (ng / mg) and radiation uptake in major organs and tumor tissues (%ID / g) [Table 4] Example 11

[0111] Ch3F8-MMAE maintains similar binding affinity to ch3F8. Ch3F8-MMAE binding affinity to HER3 was measured by FACS using SP2 / 0-HER3. The results (Figure 9) showed that ch3F8-MMAE has the same binding affinity to SP2 / 0-HER3 as ch3F8, and hardly binds to HER3-negative SP2 / 0. Example 12

[0112] Cytotoxicity of ch3F8-MMAE. The cytotoxicity of ch3F8-MMAE was measured in multiple cell lines using ATPlite. Cells were treated with various concentrations of ch3F8-MMAE for 5 days. The cytotoxicity of ch3F8-MMAE was measured with ATPlite. The data (Figure 10) showed that ch3F8-MMAE has strong cytotoxicity in killing 7901, HT-29, MCF-7, N87, MDA-MB-453, SK-BR-3, and SP2 / 0-HER3 HER3(+) cells, but has little effect on HER3(-) cells SP2 / 0-WT. The cytotoxicity IC 50 are shown in Table 5. Data were analyzed with GraphPad Prism 6.0. [Table 5] Example 13

[0113] Ch3F8-MMAE inhibits tumor growth in the gastric model GAS078. Ch3F8-MMAE was administered intravenously at 3 mg / kg once a week for 3 weeks, and the tumor suppression effect was measured every 3-4 days and continued for 10 days after the last administration. Antibody 3F8 was administered in parallel, and saline was used as a vehicle control. The results are shown in Figure 11. Example 14

[0114] Binding affinity of humanized 3F8 (hu3F8) and chimeric 3F8. The binding kinetics of three clones of humanized 3F8 and chimeric 3F8 were measured by Biacore. The three humanized 3F8 clones (clone 1, clone 2, and clone 3) have different heavy chains while sharing the same heavy chain CDRs and one light chain. The amino acid sequences of the heavy and light chains of the three humanized 3F8 clones and their coding DNA sequences are listed below. Unless otherwise stated, clone 3 was used as hu3F8 in the following examples.

[0115] All data listed in Table 6 was processed using Biacore T200 Evaluation software version 3.1. [Table 6] Example 15

[0116] Three clones of hu3F8 were incubated at pH 3.5 for 0, 2, 4 and 6 hours and then proceeded to ELISA assay to measure binding affinity. The results (Figure 12A) showed that acid treatment had little effect on binding affinity.

[0117] EC of three clones of hu3F8 after incubation at pH 3.5 for 0, 2, 4, and 6 hours 50 . E.C. 50 was measured by ELISA. The results (Table 7) showed that acid treatment had little effect on the binding affinity. [Table 7]

[0118] The three clones of hu3F8 were incubated at 40° C. for various days and then subjected to ELISA assay to measure binding affinity. The chimeric antibodies were measured in parallel. The results are shown in FIG. 12B.

[0119] EC of three clones of hu3F8 incubated at 40°C for various days. 50 Three clones of humanized 3F8 antibody were incubated in saline at 40°C for 7 or 14 days, and the binding affinity after this heat stress test was measured by ELISA. Chimeric 3F8 was measured in parallel. The results (Table 8) showed that heat stress had little effect on the binding affinity. [Table 8]

[0120] Three clones of hu3F8 were freeze-thawed for 3 or 5 cycles and then subjected to ELISA assay to measure binding affinity. Chimeric antibodies were measured in parallel. The results (Figure 12C) showed that repeated freeze-thawing had little effect on binding affinity.

[0121] EC of three clones of hu3F8 subjected to multiple cycles of freeze-thaw stress test 50 are also shown in Table 9. [Table 9]

[0122] Taken together, these data indicate that hu3F8 maintains binding affinity even after heat, acid, and repeated freeze-thaw stress testing, indicating that hu3F8 has a favorable developability profile. Example 16

[0123] Aggregation assessment of three clones of hu3F8 after stress test and their counterparts stored at 4° C. Aggregation of the three hu3F8 antibodies after acid treatment, repeated freeze-thawing, and incubation at 4° C. was measured by SEC-HPLC and compared to each antibody stored at 4° C. The results are shown in FIG. 10. After stress test, all clones maintained more than 95% monomer, suggesting little tendency for aggregation. [Table 10] Example 17

[0124] PTM (post-translational modification) analysis of hu3F8. P1: hu3F8 unstressed. P2: hu3F8, stressed at 40°C for 2 weeks. Hu3F8 unstressed or stressed at 40°C for 2 weeks was digested with trypsin and subsequently analyzed for post-translational modifications by mass spectrometry. The data (Table 11) showed a slight increase in deamidation at HC:372-393. In general, there were no significant differences in PTMs before and after stress, suggesting a good developability profile. [Table 11] Example 18

[0125] hu3F8-MMAE dose-dependently inhibits tumor growth in the gastric PDX model GAS078. Hu3F8-MMAE was administered intravenously at 1, 3 mg / kg once a week for 4 weeks. U3-1402 was administered in parallel at 10 mg / kg. Tumor size and body weight were monitored every 3-4 days. The data show that hu3F8-MMAE dose-dependently inhibits tumor growth (Figure 13A) and has little effect on body weight (Figure 13B). Hu3F8-MMAE at 3 mg / kg is comparable to U3-1402 at 10 mg / kg in terms of tumor inhibition. Example 19

[0126] hu3F8-MMAE inhibits tumor growth in the gastric PDX model GAS078 with a single injection at 10 mg / kg. U3-1402, an ADC that targets HER3 via the anti-HER3 antibody patritumab, was administered in parallel. Both hu3F8-MMAE and U3-1402 were administered once at 10 mg / kg and tumor inhibition was monitored every 3-4 days. The data (Figure 14) showed that hu3F8-MMAE at 10 mg / kg was more potent than U3-1402 at 10 mg / kg in inhibiting tumor growth in the GAS078 model. Example 20

[0127] hu3F8-MMAE inhibits tumor growth in the gastric PDX model GAS078 at 6 mg / kg. U3-1402 is an ADC that targets HER3 via the anti-HER3 antibody patritumab. Hu3F8-MMAE was administered intravenously at 6 mg / kg once a week for three weeks. Patritumab, the anti-HER3 antibody portion of U3-1402, and U3-1402 were administered in parallel at 10 mg / kg once a week for three weeks. Observations continued until day 37, 16 days after the final administration. Tumor size and body weight were monitored every 3-4 days. The data (Figure 15) show that hu3F8-MMAE inhibits tumor growth with comparable potency to U3-1402 during drug administration, but with more sustained efficacy. Example 21

[0128] hu3F8-MMAE inhibits tumor growth in colon PDX model CS226. Three groups of animals were treated with vehicle, 10 mg / kg U3-1402, and 3 mg / kg hu3F8-MMAE, respectively, for three weeks. Tumor inhibition was measured every three days. Both U3-1402 and hu3F8-MMAE significantly inhibit tumor growth compared to the vehicle-treated group. Furthermore, 3 mg / kg hu3F8 is non-inferior to 10 mg / kg U3-1402 (Figure 16).

[0129] Here, we report a new anti-HER3 antibody or ADC molecule that efficiently inhibits tumor cell proliferation in vitro and in vivo with a reasonable safety margin. A HER3 antibody, 3F8, was identified from a mouse hybridoma immunized with SP2 / 0 cells overexpressing human HER3. 3F8 recognizes human and monkey HER3 with subnanomolar binding affinity and high selectivity over other ERBB family members. Moreover, it is rapidly and efficiently taken up by cells with various levels of HER3, a property considered essential for a desirable ADC drug. PET imaging studies showed that [89Zr]Zr-3F8 significantly accumulated in tumors in PDX models, indicating that 3F8 may be a highly efficient vehicle to deliver cytotoxicity to tumor cells. Therefore, MMAE-conjugated 3F8 was generated and tested for tumor inhibitory effects. In vitro evaluation showed that 3F8-MMAE inhibited HER3-expressing tumor cells susceptible to sensitivity to MMAE with an IC of approximately 1 nM. 50 3F8-MMAE selectively kills HER3-negative cells while leaving HER3-negative cells intact. Efficacy studies showed that 3F8-MMAE inhibited tumor growth in a dose-dependent manner with no significant effect on body weight or observable hematologic toxicity. 3F8, hu3F8, and the humanized version of hu3F8-MMAE maintain the above-mentioned binding affinity, selectivity, and tumor inhibition characteristics comparable to U3-1402 in PDX models. Hu3F8 has a favorable developability profile with little change in binding affinity, aggregation, and post-translational modifications upon stress testing of repeated freeze-thaw treatments, acid incubation, and storage at 40°C. In summary, 3F8 or its ADC hu3F8-MMAE is a promising tumor treatment approach and may provide an alternative to unmet medical needs amid the growing challenge of drug resistance.

[0130] Some amino acid and nucleic acid sequences are shown below. Numbering of antibody sequences is based on Kabat. Mouse 3F8 light chain variable region: Nucleotide acid sequence: ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGCACTTTTAA ACTCCTGATCTACTACACATCAATATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAGGATATTGCCACTTACTTTTGCCAACAGGGTGATACGCTTCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 1) Amino acid sequence: MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTFKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTLPPTFGAGTKLELK (SEQ ID NO: 2) [Table 12] Mouse 3F8 heavy chain variable region: Nucleotide sequence: ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACAACTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGCTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAGACAAACTGGAATGGATGGGCTACA TAAGCTACGACGGTCGCAATAATTTCAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTTCTGGGACACAGCTACATATTACTGTGCAAGAGATGGGGATTACGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 11) Amino acid sequence: MKVLSLLYLLTAIPGILSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGDKLEWMGYISYDGRNNFNPSLKNRISITRDTSKNQFFLKLNSVTSGDTATYYCARDGDYDYFDYWGQGTTLTVSS (SEQ ID NO: 12) [Table 13] Amino acid sequence of humanized 3F8 heavy chain: (clone 3) MGWSCIILFLVATATGVHSQVQLQESGPGLVKPSETLSLTTCTVSGYSITSAYYWNWIRQPFGKGLEWMGYISYDGRNNFNPSLKNRVSISRDTSKNQFSLKLSSVTAADTATYYCARD GDYDYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21) Nucleotide sequence of humanized 3F8 heavy chain: (clone 3) Amino acid sequence of humanized 3F8 light chain: (Clones 1-3) MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGDTLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) Nucleotide sequence of humanized 3F8 light chain: (Clones 1-3) ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGTGATATTCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGATAGAGTGACCATCACATGTCGGGCCTCTCAGGACATCAGCAACTACCTGAACTGGTATCAGCAAAAGCCCGGCAAAGCCCCTAAGCTGCTGATCTACTACACCAGCATCCTGCACAGCGGAGTGCCATCTAGATTCAGCGGCTCTGGCAGCGGCACCGACTACACATTTACCATCTCCTCCCTCCAGCCTGAGGACATCGCTACATACTTCTGCCAGCAGGGCGACACCCTGCCTCCTACCTTCGGCGGCGGAACAAAGCTGGAAATCAAGAGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATGCTGATAA(SEQ ID NO: 24) Amino acid sequence of humanized 3F8 heavy chain: (Clone 1) MGWSCIILFLVATATGVHSQVQLQESGPGLVKPSETLSLTTCTVSGYSITSAYYWNWIRQPPGKGLEWIGYISYDGRNNFNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD GDYDYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25) Nucleotide sequence of humanized 3F8 heavy chain: (clone 1) Amino acid sequence of humanized 3F8 heavy chain: (clone 2) MGWSCIILFLVATATGVHSQVQLQESGPGLVKPSETLSLTTCTVSGYSITSAYYWNWIRQPFGKGLEWMGYISYDGRNNFNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARD GDYDYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27) Nucleotide sequence of humanized 3F8 heavy chain: (clone 2) References: Brian M. Zeglis and Jason S. Lewis. The bioconjugation and radiosynthesis of 89 Zr-DFO-labeled antibodies. J. Vis. Exp. 2015; (96): 52521. Kuramochi T et al. Humanization and simultaneous optimization of monoclonal antibody. Methods Mol Biol. 2014; 1060: 123 - 37.

Claims

1. An anti-HER3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, an anti-HER3 antibody or antigen-binding fragment thereof.

2. The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the VH comprises CDR-H1 of SEQ ID NO: 15, CDR-H2 of SEQ ID NO: 17, and CDR-H3 of SEQ ID NO:

19.

3. Further comprising a light chain variable region (VL), wherein the VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, the anti-HER3 antibody or antigen-binding fragment according to claim 1.

4. The anti-HER3 antibody or antigen-binding fragment according to claim 3, wherein the VL comprises CDR-L1 of SEQ ID NO: 5, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO:

9.

5. The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 12 with or without the leader sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

6. The anti-HER3 antibody or antigen-binding fragment according to claim 3, wherein the VL comprises the amino acid sequence of SEQ ID NO: 2 with or without the leader sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

7. The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

8. The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the heavy chain of the antibody is of the IgG1 type.

9. The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the antibody is a humanized antibody, and the heavy chain has the amino acid sequence of SEQ ID NO: 21, 25, or 27, with or without a leader sequence included therein, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those.

10. The anti-HER3 antibody or antigen-binding fragment according to claim 9, wherein the leader sequence contains the amino acids at positions 1 to 19 of the amino acid sequence of SEQ ID NO: 21, 25, or 27.

11. The anti-HER3 antibody or antigen-binding fragment according to claim 10, wherein the antibody is a humanized antibody, and the heavy chain contains the amino acids at positions 20 to 467 of SEQ ID NO: 21, 25, or 27.

12. The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the antibody is a humanized antibody, and the light chain has the amino acid sequence of SEQ ID NO: 23, with or without a leader sequence included therein, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that.

13. The anti-HER3 antibody or antigen-binding fragment according to claim 12, wherein the leader sequence contains the amino acids at positions 1 to 19 of the amino acid sequence of SEQ ID NO:

23.

14. The anti-HER3 antibody or antigen-binding fragment according to claim 13, wherein the antibody is a humanized antibody, and the light chain contains the amino acids at positions 20 to 233 of SEQ ID NO:

23. **Claim 15**: The anti-HER3 antibody or antigen-binding fragment according to claim 1, wherein the VH comprises a VH sequence comprising the sequence of SEQ ID NO: 21, 25 or 27. **Claim 16**: The anti-HER3 antibody or antigen-binding fragment according to claim 15, wherein the VH comprises the amino acids at positions 20 to 137 of the amino acid sequence of SEQ ID NO: 21, 25 or 27. **Claim 17**: The anti-HER3 antibody or antigen-binding fragment according to claim 3, wherein the VL comprises a VL sequence comprising the sequence of SEQ ID NO:

23. **Claim 18**: The anti-HER3 antibody or antigen-binding fragment according to claim 17, wherein the VL comprises the amino acids at positions 20 to 126 of the amino acid sequence of SEQ ID NO:

23. **Claim 19**: (a) The HER3 is human or monkey HER3, (b) The anti-HER3 antibody inhibits the phosphorylation of the HER3 induced by NRG1, (c) The anti-HER3 antibody binds to human or monkey HER3 with an EC50 of less than 1 nM, and / or (d) The anti-HER3 antibody has internalization activity when binding to the HER3, The anti-HER3 antibody or antigen-binding fragment according to any one of claims 1 to 18. **Claim 20** An isolated nucleic acid comprising a polynucleotide sequence encoding the anti-HER3 antibody or antigen-binding fragment according to any one of claims 1 to 18. **Claim 21** A vector comprising the isolated nucleic acid according to claim 20. **Claim 22** A host cell comprising the isolated nucleic acid according to claim 20 or a vector comprising the isolated nucleic acid. **Claim 23**: An antibody conjugate comprising the anti-HER3 antibody or antigen-binding fragment according to any one of claims 1 to 18 bound to a chemical moiety, wherein the antibody conjugate is an antibody-drug conjugate (ADC). **Claim 24** The antibody conjugate according to claim 23, wherein the anti-HER3 antibody or antigen-binding fragment is bound to the chemical moiety via a linker. **Claim 25** The antibody conjugate according to claim 23, wherein the chemical moiety is a radioisotope, a chemotherapeutic agent, or a cytotoxic drug. **Claim 26** The antibody conjugate according to claim 25, wherein the cytotoxic drug is selected from auristatin E, auristatin F, MMAE, and MMAF. **Claim 27** A pharmaceutical composition comprising an anti-HER3 antibody or antigen-binding fragment according to any one of claims 1 to 18, or an antibody conjugate comprising an anti-HER3 antibody or antigen-binding fragment bound to a chemical moiety, and a pharmaceutically acceptable carrier. **Claim 28** Use of an antibody-drug conjugate comprising an anti-HER3 antibody or antigen-binding fragment according to any one of claims 1 to 18, or an anti-HER3 antibody or antigen-binding fragment bound to a chemical moiety, in the manufacture of a medicament for the treatment of cancer, wherein the cancer expresses HER3. **Claim 29** The use according to claim 28, wherein the cancer is gastric cancer or colorectal cancer. **Claim 30** A pharmaceutical composition for use in the treatment of cancer, comprising an antibody-drug conjugate comprising an anti-HER3 antibody or antigen-binding fragment according to any one of claims 1 to 18, or an anti-HER3 antibody or antigen-binding fragment bound to a chemical moiety, wherein the cancer expresses HER3. **Claim 31** The pharmaceutical composition according to claim 30, wherein the cancer is gastric cancer or colorectal cancer.

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