Anti-her2 / Anti-4-1BB bispecific antibody for treatment of biliary tract cancer

Abstract

Disclosed are a pharmaceutical composition and a therapeutic method that can exhibit an effective antitumor effect and safety by administering an anti-HER2 / anti-4-1BB bispecific antibody to a patient with biliary tract cancer.

Description

ANTI-HER2 / ANTI-4-1BB BISPECIFIC ANTIBODY FOR TREATMENT OF BILIARY TRACT CANCER

[0001] The present invention relates to a pharmaceutical composition containing an anti-HER2 / anti-4-1BB bispecific antibody, which may be used for the treatment of biliary tract cancer, and a method for treating biliary tract cancer using the same.

[0002] 4-1BB protein is a member of the TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule which is expressed after activation of both innate and adaptive immune cells. 4-1BB plays an important role in modulating the activities of various immune cells. 4-1BB agonists enhance the proliferation and survival of immune cells, and the secretion of cytokines, and activate cytolytic CD8 T cells. Many other studies have shown that activation of 4-1BB enhances the immune response to eliminate tumors in mice. Therefore, it suggests that 4-1BB is a promising target molecule in cancer immunology. Despite its antitumor efficacy, the previously developed first-generation anti-4-1BB antibody (urelumab) caused severe liver toxicity in clinical applications.

[0003] HER2 protein is a member of the epidermal growth factor receptor (EGFR) family and is involved in various mechanisms related to tumors. HER2 is a representative receptor tyrosine kinase (RTK) present on the cell surface and induces proliferation and invasion of cancer cells, angiogenesis, etc. Representative HER2-targeted therapeutic agents developed to date include trastuzumab, pertuzumab, etc.

[0004] Meanwhile, biliary tract cancer is a cancer that occurs in the bile duct, which is a tube that carries bile produced in the liver to the duodenum. Bile is produced in liver cells, flows out of the liver, and is discharged through the papilla of the duodenum. The bile ducts are divided into the intrahepatic bile ducts that are located within the liver and the extrahepatic bile ducts that extend from the liver to the duodenum. Biliary tract cancer, a cancer that occurs in the bile duct, is a tumor composed of cells that resemble the epithelium of the bile duct, and is divided into intrahepatic biliary tract cancer and extrahepatic biliary tract cancer based on the anatomical location of occurrence. Intrahepatic biliary tract cancer is subdivided into peripheral biliary tract cancer and hilar biliary tract cancer, and extrahepatic biliary tract cancer is subdivided into upper (proximal), middle, and lower (distal) biliary tract cancers based on the location of occurrence. There are differences in clinical manifestations, treatment methods, and prognosis between intrahepatic biliary tract cancer and extrahepatic biliary tract cancer.

[0005] Since biliary tract cancer has no characteristic symptoms until it is quite advanced, it is difficult to diagnose early, and at the time of diagnosis, it often invades surrounding major organs and is not possible to radically resect. Therapeutic methods for biliary tract cancer vary depending on the location and degree of progression of the cancer. The only therapeutic method for complete cure of biliary tract cancer is surgical resection, but biliary tract cancer is often found in an advanced state at the time of diagnosis in more than two-thirds of patients, making radical resection difficult. More than 80% of biliary tract cancer patients, including those who relapse after surgery, receive chemotherapy. Currently, gemcitabine and cisplatin are known to be the most effective for biliary tract cancer, but their therapeutic effects are still insufficient compared to those for other cancers, and an effective therapeutic method for biliary tract cancer is required.

[0006] Accordingly, the present invention aims to provide an improved method of treating a subject with biliary tract cancer using a specific antibody targeting HER2 and 4-1BB.

[0007] An object of the present invention is to provide a method capable of treating or alleviating biliary tract cancer in a subject in need thereof, and a pharmaceutical composition that may be used for the method.

[0008] However, the objects to be achieved by the present invention are not limited to the object mentioned above, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

[0009] Hereinafter, various embodiments described herein will be described. In the following description, numerous specific details are set forth, such as specific configurations, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparation techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "another embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "another embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0010] Unless otherwise defined in the specification,　all the scientific and technical terms used in the specification　have the same meanings as commonly understood　by those skilled in the technical field to which the present invention pertains.

[0011] According to one embodiment of the present invention, the present invention relates to a pharmaceutical composition for treating biliary tract cancer, containing an anti-HER2 / anti-4-1BB bispecific antibody as an active ingredient.

[0012] According to another embodiment of the present invention, the present invention relates to a method for treating biliary tract cancer, comprising a step of administering to a subject an effective amount of an anti-HER2 / anti-4-1BB bispecific antibody.

[0013] The present invention provides a method capable of effectively and safely treating biliary tract cancer by administering a specific anti-HER2 / anti-4-1BB bispecific antibody to a patient with biliary tract cancer (preferably biliary tract cancer having a specific phenotype) using a specific clinical dosage regimen, for example, at a specific dosage according to a specific dosing schedule.

[0014] Definitions

[0015] As used herein, the term "consisting of a sequence", "consisting essentially of a sequence", or "comprising a sequence" may mean any case comprising the sequence, but is not intended to exclude a case comprising further sequence other than the sequence.

[0016] The term "antibody" as used herein refers to a protein molecule acting as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a particular antigen. In the present invention, the antibodies include all of polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. In addition, the antibodies include chimeric antibodies, human antibodies, humanized antibodies, bivalent antibodies, bispecific molecules, minibodies, domain antibodies, bispecific or multispecific antibodies, immune cell-engaging bispecific or multispecific antibodies, antibody mimetics, unibodies, diabodies, triabodies, or tetrabodies. The term further includes single-chain antibodies capable of binding to neonatal Fc receptor (FcRn), scAbs (single-chain antibodies), derivatives of an antibody constant region, and artificial antibodies based on a protein scaffold.　A whole antibody has a structure of two full-length light chains (LCs) and two full-length heavy chains (HCs), in which each light chain is linked to a heavy chain by disulfide bonds. The whole antibodies include IgA, IgD, IgE, IgM and IgG, and subtypes of IgG include IgG1, IgG2, IgG3 and IgG4. As an example, the antibody provided by the present invention may be an IgG antibody.

[0017] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Typically, immunoglobulins have heavy chains and light chains, in which each of the heavy chains and light chains contains a constant region and a variable region (the regions are also known as domains). The variable regions of the light and heavy chains comprise three variable regions, called complementarity-determining regions (CDRs), and four framework regions.

[0018] As used herein, the term "complementarity-determining region" or "CDR region" or "CDR" refers to a region in an antibody variable domain that is highly variable in sequence, forms a structurally defined loop ("hypervariable loop") and / or comprises antigen-contacting residues ("antigen-contacting sites").　CDRs are primarily responsible for binding to antigen epitopes. The complementarity-determining regions are interspersed between relatively conserved regions, called framework regions (FRs). Each of the heavy chain variable region (VH) and the light chain variable region (VL) consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The CDRs of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3, the CDRs of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, the FRs of the heavy chain variable region may be referred to as HFR1, HFR2, HFR3, and HFR4, and the FRs of the light chain variable region may be referred to as LFR1, LFR2, LFR3, and LFR4. Here, the CDRs and FRs are numbered sequentially from the N-terminus. In the amino acid sequence of a given light chain variable region or heavy chain variable region, the precise amino acid sequence boundary of each CDR can be determined by any one of many well-known antibody CDR assignment systems or a combination thereof, which include, for example: Chothia based on the three-dimensional structure of an antibody and the topology of CDR loops (Chothia et, al. (1989) Nature 342:877-883, Al-Lazikani et, al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of antibody sequence (Kabat et, al, Sequences of Protein of Immunological Interest, the 4th　Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), as well as North CDR definition based on the affinity propagation clustering using a large number of crystal structures. However, the antibody sequence numbering described in the present specification is based on Kabat numbering.

[0019] The antibody of the present invention may comprise a constant region, wherein the constant region may be a constant region derived from IgG, IgA, IgD, IgE, IgM, or combinations thereof or hybrids thereof, without being limited thereto. Here, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin constant region of the same origin is linked to a single-chain polypeptide of a different origin to form a dimer or multimer. For example, a dimer or multimer may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE and IgM. Additionally, the term "hybrid" means that sequences corresponding to two or more immunoglobulin heavy-chain constant regions of different origins are present in a single-chain immunoglobulin heavy-chain constant region. For example, a domain hybrid may be composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG, IgA, IgD, IgE and IgM.

[0020] As used herein, the term "a full-length antibody" refers to an antibody has a structure of two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. Full-length antibodies include IgA, IgD, IgE, IgM, and IgG, and subtypes of IgG include IgG1, IgG2, IgG3, and IgG4.

[0021] As used herein, the terms "fragment", "binding fragment of a polypeptide" and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv or scFv. The term "Fd" refers to the heavy chain portion contained in the Fab fragment. As used herein, the term "Fab" generally refers to a fragment comprising a heavy chain variable domain and a light chain variable domain, and further comprising a light chain constant domain and a heavy chain first constant domain (CH1). The term "Fab'" generally refers to a fragment which differs from a Fab by the addition of a few residues (including one or more cysteines from the antibody hinge region) to the carboxyl terminus of the heavy chain CH1 domain. The term "F(ab')2" generally refers to a dimeric antibody fragment of Fab', which comprises two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fv" generally refers to the minimum antibody fragment that contains a complete antigen recognition and binding site. In certain cases, the fragment may consist of a dimer of a heavy chain variable region and a light chain variable region that are closely non-covalently associated. The term "dsFv" generally refers to a disulfide-stabilized Fv fragment in which the bond between the single light chain variable region and the single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In the present invention, the term "scFv" generally refers to a monovalent molecule produced by covalently linking and pairing of a heavy chain variable domain with a light chain variable domain of an antibody by a flexible peptide linker. Such a scFv molecule may have the following general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0022] As used herein, the term "about" is intended to refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurements. For example, the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes, but is not limited to, a variation of ±1 in the most precise significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values that are within 10-fold, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. The numerical quantities include, but are not limited to, all numbers within an equivalent or similar range.

[0023] As used herein, the term "tumor burden" also referred to as "tumor load", refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumor(s), throughout the body, including lymph nodes and bone narrow. As used herein, the term "tumor size" refers to the total size of the tumor which can be measured as the length and width of a tumor. Tumor burden and tumor size may be determined by a variety of methods known in the art, for example, by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scans.

[0024] As used herein, the term "advanced" cancer refers to one which has spread outside the site or organ of origin, either by local invasion or metastasis. Accordingly, the term "advanced" cancer includes both locally advanced and metastatic disease.

[0025] As used herein, the term "metastatic" cancer refers to cancer that has spread from one part of the body (e.g., the biliary tract) to another part of the body.

[0026] As used herein, the term "recurrent" cancer refers to cancer which has regrown, either at the initial site or at a distant site, after a response to initial therapy, such as surgery.

[0027] As used herein, the term "non-resectable" or "unresectable" cancer refers to cancer which is not able to be removed (resected) by surgery.

[0028] Anti-HER2 / Anti-4-1BB Bispecific Antibody

[0029] In the present invention, the anti-HER2 / anti-4-1BB bispecific antibody comprises an anti-4-1BB targeting moiety and an anti-HER2 targeting moiety.

[0030] Anti-4-1BB Targeting Moiety

[0031] In the present invention, the anti-4-1BB targeting moiety may comprise an anti-4-1BB antibody or an antigen-binding fragment thereof.

[0032] As used herein, the term "4-1BB" refers to a member of the TNF-receptor superfamily (TNFRSF), which is a co-stimulatory molecule which is expressed after activation of immune cells, both innate and adaptive immune cells, and is also called CD137 or TNFRSF9 (TNF receptor superfamily member 9). 4-1BB plays an important role in modulating the activities of various immune cells. As used herein, 4-1BB may be derived from a mammal, for example,Homo sapiens(human) (NCBI Accession No. NP_001552.2). Also, for example, human 4-1BB protein (NP_001552.2) may be represented by the amino acid sequence (SEQ ID NO: 1).

[0033] In the present invention, the anti-4-1BB antibody or an antigen-binding fragment thereof may comprise: a heavy chain variable region comprising HCDR1 represented by SEQ ID NO: 2, HCDR2 represented by SEQ ID NO: 3, and HCDR3 represented by SEQ ID NO: 4; and a light chain variable region comprising LCDR1 represented by SEQ ID NO: 5, LCDR2 represented by SEQ ID NO: 6, and LCDR3 represented by SEQ ID NO: 7.

[0034] In the present invention, the amino acid sequences of the CDRs (complementarity determining regions) of the anti-4-1BB antibody or an antigen-binding fragment thereof are exemplified in Table 1 below.

[0035] SEQ ID NO.NameAmino acid sequence information2HCDR1SYDMS3HCDR2WISYSGGSIYYADSVKG4HCDR3DAQRNSMREFDY5LCDR1SGSSSNIGNNYVT6LCDR2ADSHRPS7LCDR3ATWDYSLSGYV

[0036] In addition, in the present invention, the anti-4-1BB antibody or an antigen-binding fragment thereof may comprise: a heavy chain variable region comprising HFR1 represented by SEQ ID NO: 8, HFR2 represented by SEQ ID NO: 9, HFR3 represented by SEQ ID NO: 10, and HFR4 represented by SEQ ID NO: 11; and a light chain variable region comprising LFR1 represented by SEQ ID NO: 12, LFR2 represented by SEQ ID NO: 13, LFR3 represented by SEQ ID NO: 14, and LFR4 represented by SEQ ID NO: 15.

[0037] In the present invention, the amino acid sequences of the FRs (frameworks) of the anti-4-1BB antibody or an antigen-binding fragment thereof are exemplified in Table 2 below.

[0038] SEQ ID NO.NameAmino acid sequence information8HFR1EVQLLESGGGLVQPGGSLRLSCAASGFTFS9HFR2WVRQAPGKCLEWVS10HFR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR11HFR4WGQGTLVTVSS12LFR1QSVLTQPPSASGTPGQRVTISC13LFR2WYQQLPGTAPKLLIY14LFR3GVPDRFSGSKSGTSASLAISGLRSEDEADYYC15LFR4FGCGTKLTVL

[0039] In the present invention, the anti-4-1BB antibody or an antigen-binding fragment thereof may comprise a heavy chain variable region represented by SEQ ID NO: 16, and a light chain variable region represented by SEQ ID NO: 17.

[0040] In the present invention, the amino acid sequences of the VH (heavy chain variable region) and VL (light chain variable region) of the anti-4-1BB antibody or an antigen-binding fragment thereof are exemplified in Table 3 below.

[0041] SEQ ID NO.NameAmino acid sequence information16VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS17VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL

[0042] In the present invention, the antigen-binding fragment of the anti-4-1BB antibody may be a scFv (single-chain variable fragment) that specifically binds to 4-1BB.

[0043] The anti-4-1BB scFv of the present invention may comprise: a heavy chain variable region comprising HCDR1 represented by SEQ ID NO: 2, HCDR2 represented by SEQ ID NO: 3, and HCDR3 represented by SEQ ID NO: 4; and a light chain variable region comprising LCDR1 represented by SEQ ID NO: 5, LCDR2 represented by SEQ ID NO: 6, and LCDR3 represented by SEQ ID NO: 7.

[0044] The anti-4-1BB scFv of the present invention may comprise a heavy chain variable region represented by SEQ ID NO: 16, and a light chain variable region represented by SEQ ID NO: 17.

[0045] In the anti-4-1BB scFv of the present invention, the heavy chain variable region and the light chain variable region may be linked directly without a linker, or may be linked to each other via a peptide linker.

[0046] As used herein, the term "peptide linker" may refer to an oligopeptide comprising 1 to 100 amino acids, particularly 2 to 50 amino acids, each of which may be any kind of amino acid without any restrictions. Any conventional peptide linker may be used with or without an appropriate modification to comply with specific purposes. In a specific embodiment, the peptide linker may comprise, for example, Gly, Asn and / or Ser residues, and / or comprise neutral amino acids such as Thr and / or Ala. The amino acid sequences suitable for the peptide linker may be known. In the relevant art, the length of the peptide linker can be properly determined within such a limit that the functions of the polypeptide and / or scFv will not be affected. For instance, the peptide linker may consist of a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids, each of which may be independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented by (GmSl)n(where m, l, and n are the numbers of "G", "S" and "(GmSl)", respectively, and may be independently selected from an integer ranging from about 1 to about 10, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In one embodiment, the peptide linker may be a peptide of (GGGGS)2(SEQ ID NO: 30), (GGGGS)3(SEQ ID NO: 31), (GGGGS)4(SEQ ID NO: 32), or (GS)9(SEQ ID NO: 33), without being limited thereto.

[0047] The heavy chain variable region and the light chain variable region in the anti-4-1BB scFv of the present invention may be linked to each other in any order without particular limitation. As a non-limiting example, the anti-4-1BB scFv may comprise a light chain variable region and a heavy chain variable region, in the direction from the N-terminus to the C-terminus. Alternatively, the anti-4-1BB scFv may comprise a heavy chain variable region and a light chain variable region, in the direction from the N-terminus to the C-terminus. In addition, when the heavy chain variable region and the light chain variable region are linked to each other via a peptide linker, the anti-4-1BB scFv may comprise the light chain variable region, the peptide linker and the heavy chain variable region, in the direction from the N-terminus to the C-terminus, or comprise the heavy chain variable region, the peptide linker and the light chain variable region, in the direction from the N-terminus to the C-terminus.

[0048] As an example, the heavy chain variable region and the light chain variable region in the anti-4-1BB scFv of the present invention may be linked to each other by a peptide linker comprising the amino acid sequence represented by SEQ ID NO: 32.

[0049] As an example, the anti-4-1BB scFv of the present invention comprises a heavy chain variable region represented by SEQ ID NO: 16, and a light chain variable region represented by SEQ ID NO: 17, wherein the heavy chain variable region and the light chain variable region may be linked to each other by a peptide linker comprising the amino acid sequence represented by SEQ ID NO: 32.

[0050] As an example, the anti-4-1BB scFv of the present invention may comprise, in the direction from the N-terminus to the C-terminus, a heavy chain variable region represented by SEQ ID NO: 16, a peptide linker comprising an amino acid sequence represented by SEQ ID NO: 32, and a light chain variable region represented by SEQ ID NO: 17.

[0051] As an example, the anti-4-1BB scFv of the present invention may comprise, in the direction from the N-terminus to the C-terminus, a light chain variable region represented by SEQ ID NO: 17, a peptide linker comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain variable region represented by SEQ ID NO: 16.

[0052] The amino acid sequence of the anti-4-1BB scFv (single-chain variable fragment) in the present invention is exemplified in Table 4 below.

[0053] SEQ ID NO.NameAmino acid sequence information16VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS17VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL32LinkerGGGGSGGGGSGGGGSGGGGS18ScFv (VL-linker-VH)QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS

[0054] Anti-HER2 Targeting Moiety

[0055] In the present invention, the anti-HER2 targeting moiety may comprise an anti-HER2 antibody or an antigen-binding fragment thereof.

[0056] As used herein, the term "HER2 (human epidermal growth factor receptor 2)" refers to a member of the epidermal growth factor receptor (EGFR / ErbB), which is encoded by the ERBB2 gene. HER2 is known to play an essential role in regulating cell proliferation and differentiation. In particular, when HER2 binds to extracellular growth factor, it strongly tends to assemble with other HER receptors to form a homodimer and / or a heterodimer, thereby activating various signal transduction pathways, thus inducing cell death, survival, or proliferation. For example, the HER2 proteins may be polypeptides deposited under GenBank Accession Number NP_004439.2, NP_001005862.1, etc., which are encoded by the nucleotide sequences (mRNA) deposited under GenBank Accession Number NM_004448.4, NM_001005862.3, etc., respectively.

[0057] In the present invention, the anti-HER2 antibody or antigen-binding fragment thereof may be an anti-HER2 antibody selected from the group consisting of trastuzumab, pertuzumab, and trastuzumab emtansine (T-DM1), or antigen-binding fragment thereof.

[0058] In the present invention, the anti-HER2 antibody or antigen-binding fragment thereof may comprise: a heavy chain variable region comprising HCDR1 represented by SEQ ID NO: 19, HCDR2 represented by SEQ ID NO: 20, and HCDR3 represented by SEQ ID NO: 21; and a light chain variable region comprising LCDR1 represented by SEQ ID NO: 22, LCDR2 represented by SEQ ID NO: 23, and LCDR3 represented by SEQ ID NO: 24.

[0059] In the present invention, the amino acid sequences of the CDRs (complementarity determining regions) of the anti-HER2 antibody or antigen-binding fragment are exemplified in Table 5 below.

[0060] SEQ ID NO.NameAmino acid sequence information19HCDR1DTYIH20HCDR2RIYPTNGYTRYADSVKG21HCDR3WGGDGFYAMDY22LCDR1RASQDVNTAVA23LCDR2SASFLYS24LCDR3QQHYTTPPT

[0061] In the present invention, the anti-HER2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region represented by SEQ ID NO: 25, and a light chain variable region represented by SEQ ID NO: 26.

[0062] In the present invention, the amino acid sequences of the VH (heavy chain variable region) and VL (light chain variable region) of the anti-HER2 antibody or antigen-binding fragment are exemplified in Table 6 below.

[0063] SEQ ID NO.NameAmino acid sequence information25VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS26VLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0064] In the present invention, the anti-HER2 antibody or antigen-binding fragment thereof may comprise a heavy chain represented by SEQ ID NO: 27, and a light chain represented by SEQ ID NO: 28.

[0065] In the present invention, the amino acid sequences of the heavy and light chains of the anti-HER2 antibody or antigen-binding fragment in the present invention are exemplified in Table 7 below.

[0066] SEQ ID NONameAmino acid sequence information27Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK28Light chainDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0067] In the present invention, the antigen-binding fragment of the anti-HER2 antibody may be a scFv (single-chain variable fragment) that specifically binds to HER2. The anti-HER2 scFv of the present invention may comprise: a heavy chain variable region comprising HCDR1 represented by SEQ ID NO: 19, HCDR2 represented by SEQ ID NO: 20, and HCDR3 represented by SEQ ID NO: 21; and a light chain variable region comprising LCDR1 represented by SEQ ID NO: 22, LCDR2 represented by SEQ ID NO: 23, and LCDR3 represented by SEQ ID NO: 24.

[0068] The anti-HER2 scFv of the present invention may comprise a heavy chain variable region represented by SEQ ID NO: 25, and a light chain variable region represented by SEQ ID NO: 26.

[0069] In the anti-HER2 scFv of the present invention, the heavy chain variable region and the light chain variable region may be linked directly without a linker, or may be linked to each other via a peptide linker. Here, the peptide linker may consist of a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids, each of which may be independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented by (GmSl)n(where m, l, and n are the numbers of "G", "S" and "(GmSl)", respectively, and may be independently selected from an integer ranging from about 1 to about 10, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In one embodiment, the peptide linker may be a peptide of (GGGGS)2(SEQ ID NO: 30), (GGGGS)3(SEQ ID NO: 31), (GGGGS)4(SEQ ID NO: 32), or (GS)9(SEQ ID NO: 33), without being limited thereto.

[0070] The heavy chain variable region and the light chain variable region in the anti-HER2 scFv of the present invention may be linked to each other in any order without particular limitation. As a non-limiting example, the anti-HER2 scFv may comprise a light chain variable region and a heavy chain variable region, in the direction from the N-terminus to the C-terminus. Alternatively, the anti-HER2 scFv may comprise a heavy chain variable region and a light chain variable region, in the direction from the N-terminus to the C-terminus. In addition, when the heavy chain variable region and the light chain variable region are linked to each other via a peptide linker, the anti-HER2 scFv may comprise the light chain variable region, the peptide linker and the heavy chain variable region, in the direction from the N-terminus to the C-terminus, or comprise the heavy chain variable region, the peptide linker and the light chain variable region, in the direction from the N-terminus to the C-terminus.

[0071] As an example, the heavy chain variable region and the light chain variable region in the anti-HER2 scFv of the present invention may be linked to each other by a peptide linker comprising the amino acid sequence represented by any one of SEQ ID NOs: 30 to 33.

[0072] As an example, the anti-HER2 scFv of the present invention comprises a heavy chain variable region represented by SEQ ID NO: 25, and a light chain variable region represented by SEQ ID NO: 26, wherein the heavy chain variable region and the light chain variable region may be linked to each other by the peptide linker.

[0073] As an example, the anti-HER2 scFv of the present invention may comprise, in the direction from the N-terminus to the C-terminus, a heavy chain variable region represented by SEQ ID NO: 25, a peptide linker, and a light chain variable region represented by SEQ ID NO: 26.

[0074] As an example, the anti-HER2 scFv of the present invention may comprise, in the direction from the N-terminus to the C-terminus, a light chain variable region represented by SEQ ID NO: 26, a peptide linker, and a heavy chain variable region represented by SEQ ID NO: 25.

[0075] Bispecific Antibody

[0076] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise: as a HER2 targeting moiety, an anti-HER2 antibody or an antigen-binding fragment thereof capable of specifically recognizing and / or binding to a HER2 protein; and as a 4-1BB targeting moiety, an anti-4-1BB antibody or an antigen-binding fragment thereof capable of specifically recognizing and / or binding to a 4-1BB protein.

[0077] In the anti-HER2 / anti-4-1BB bispecific antibody of the present invention, the HER2 targeting moiety and the 4-1BB targeting moiety may be linked to each other directly or via a peptide linker.

[0078] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise a full-length anti-HER2 antibody and an antigen-binding fragment (e.g., scFv) of an anti-4-1BB antibody. In this case, the full-length anti-HER2 antibody and the antigen-binding fragment of the anti-4-1BB antibody may be linked to each other directly or via a peptide linker. Specifically, the N-terminus or C-terminus of the antigen-binding fragment of the anti-4-1BB antibody may be linked directly or via a peptide linker to the N-terminus and / or C-terminus of the full-length anti-HER2 antibody.

[0079] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, in the direction from the N-terminus to the C-terminus, a first polypeptide comprising a heavy chain of an anti-HER2 antibody and an anti-4-1BB scFv, and a second polypeptide comprising a light chain of an anti-HER2 antibody, without being limited thereto. Here, the anti-4-1BB scFv comprises a heavy chain variable region and a light chain variable region of the anti-4-1BB antibody, which are linked to each other in any order without particular limitation.

[0080] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, in the N-terminal to C-terminal direction, a first polypeptide comprising a heavy chain of an anti-HER2 antibody, a light chain variable region of an anti-4-1BB antibody, and a heavy chain variable region of the anti-4-1BB antibody, and a second polypeptide comprising a light chain of the anti-HER2 antibody, without being limited thereto.

[0081] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, in the N-terminal to C-terminal direction, a first polypeptide comprising a heavy chain of an anti-HER2 antibody, a heavy chain variable region of an anti-4-1BB antibody, and a light chain variable region of the anti-4-1BB antibody, and a second polypeptide comprising a light chain of the anti-HER2 antibody, without being limited thereto.

[0082] In the anti-HER2 / anti-4-1BB bispecific antibody of the present invention, the heavy chain of the anti-HER2 antibody and the anti-4-1BB scFv may be linked to each other directly or via a peptide linker (hereinafter referred to as "first peptide linker").

[0083] In addition, in the anti-HER2 / anti-4-1BB bispecific antibody of the present invention, the heavy chain variable region and the light chain variable region of the anti-4-1BB antibody in the anti-4-1BB scFv may be linked to each other directly or via a peptide linker (hereinafter referred to as "second peptide linker").

[0084] In the present invention, each of the first peptide linker and the second peptide linker may consist of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids, each of which may be independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the first peptide linker and the second peptide linker may be each independently represented by (GmSl)n(where m, l, and n are the numbers of "G", "S" and "(GmSl)", respectively, and may be independently selected from an integer ranging from about 1 to about 10, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), without being limited thereto. In one embodiment, the first peptide linker and the second peptide linker may be each independently a peptide of (GGGGS)2(SEQ ID NO: 30), (GGGGS)3(SEQ ID NO: 31), (GGGGS)4(SEQ ID NO: 32), or (GS)9(SEQ ID NO: 33), without being limited thereto.

[0085] When the anti-HER2 / anti-4-1BB bispecific antibody of the present invention comprises both the first peptide linker and the second peptide linker, the linkers may be the same as or different from each other.

[0086] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, an anti-4-1BB antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0087] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, an anti-4-1BB antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0088] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, a peptide linker comprising the amino acid sequence of SEQ ID NO: 30, 31 or 32, an anti-4-1BB antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0089] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, a peptide linker comprising the amino acid sequence of SEQ ID NO: 30, 31 or 32, an anti-4-1BB antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0090] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, a peptide linker comprising the amino acid sequence of SEQ ID NO: 32, an anti-4-1BB antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0091] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16, a peptide linker comprising the amino acid sequence of SEQ ID NO: 32, an anti-4-1BB antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0092] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB scFv comprising the amino acid sequence of SEQ ID NO: 18, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0093] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, a peptide linker comprising the amino acid sequence of SEQ ID NO: 33, an anti-4-1BB scFv comprising the amino acid sequence of SEQ ID NO: 18, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.

[0094] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may comprise, but is not limited to, a first polypeptide comprising the amino acid sequence of SEQ ID NO: 29, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 28.

[0095] In the present invention, the amino acid sequences of the first polypeptide and the second polypeptide of the anti-HER2 / anti-4-1BB bispecific antibody are exemplified in Table 8 or 9 below.

[0096] ClassificationAmino acid sequence information (N-terminus →C-terminus)First polypeptide① Anti-HER2 antibody heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27)② Peptide linkerGSGSGSGSGSGSGSGSGS (SEQ ID NO: 33)③ Anti-4-1BB scFvVLQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL (SEQ ID NO: 17)Peptide linkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32)VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS (SEQ ID NO: 16)Heavy component (①+②+③)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS (SEQ ID NO: 29)Second polypeptideAnti-HER2 antibody light chainDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28)

[0097] ClassificationAmino acid sequence information (N-terminus → C-terminus)First polypeptide(Anti-HER2 antibody heavy chain)-(peptide linker)-(anti-4-1BB scFv)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS (SEQ ID NO: 29)Second polypeptideAnti-HER2 antibody light chainDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28)

[0098] Pharmaceutical Composition

[0099] In the present invention, the anti-HER2 / anti-4-1BB bispecific antibody may be administered to a subject in need of treatment in the form of a pharmaceutical composition.

[0100] The pharmaceutical composition of the present invention may contain, in addition to the anti-HER2 / anti-4-1BB bispecific antibody as an active ingredient, a pharmaceutically acceptable carrier, lubricant, diluent, excipient, stabilizer, or the like. Examples of suitable carriers, lubricants, diluents, excipients or stabilizers include, but are not limited to, buffers such as phosphate, citrate, histidine, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, sucrose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; pH adjusting agents; and / or nonionic surfactants such as TWEENTM(polysorbate such as polysorbate 80, polysorbate 20 or the like), polyethylene glycol (PEG), and PLURONICSTM.

[0101] For delivery, the anti-HER2 / anti-4-1BB bispecific antibody, the active ingredient in the pharmaceutical composition of the present invention, may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.

[0102] The pharmaceutical composition of the present invention may be prepared as a sustained-release preparation. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, in which the matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release polypeptides for shorter time periods.

[0103] The pharmaceutical composition of the present invention is intended forin vivoadministration and may be sterilized. This is easily achieved by filtration through a sterile filtration membrane.

[0104] The pharmaceutical compositions of the present invention may be in any suitable form, for example, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders, to name just a few non-limiting alternatives. Such compositions (or formulations) may be prepared using methods known in the art, such as conventional dissolution and mixing procedures.

[0105] As an example, the pharmaceutical composition of the present invention may be prepared in the form of a lyophilized formulation, milled powder, or an aqueous solution, without being limited thereto.

[0106] Subject

[0107] The present invention provides a clinical method for treating biliary tract cancer in a subject using the above-described anti-HER2 / anti-4-1BB bispecific antibody.

[0108] As used herein, the term "subject" includes any individual who has one or more symptoms or signs of biliary tract cancer, has been diagnosed as having developed biliary tract cancer, or is predicted to have a high probability of developing biliary tract cancer.

[0109] The subject may include both a human and a non-human animal. The term "non-human animal" may include all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

[0110] As used herein, the term "biliary tract cancer (BTC)" refers to a solid carcinoma, also called "bile duct cancer", and specifically includes gallbladder cancer, ampullary carcinoma, and cholangiocarcinoma. Here, the cholangiocarcinoma (CCA) may also be classified as intrahepatic CCA or extrahepatic CCA.

[0111] As an example, the biliary tract cancer in the present invention may be gallbladder cancer.

[0112] As an example, the biliary tract cancer in the present invention may be ampullary carcinoma (for example, ampullary adenocarcinoma).

[0113] As an example, the biliary tract cancer in the present invention may be cholangiocarcinoma.

[0114] As an example, the biliary tract cancer in the present invention may be intrahepatic cholangiocarcinoma.

[0115] As an example, the biliary tract cancer in the present invention may be extrahepatic cholangiocarcinoma.

[0116] As an example, the biliary tract cancer in the present invention may include at least one of gallbladder cancer, ampullary carcinoma, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma.

[0117] In the present invention, the biliary tract cancer may be HER2-positive biliary tract cancer. As used herein, the term "HER2-positive biliary tract cancer" refers to biliary tract cancer which is classified as a HER2+ tumor by immunohistochemistry (IHC) orin situhybridization (ISH) analysis. In the present invention, the HER2-positive biliary tract cancer may be (i) biliary tract cancer given a score of 3+ for HER2 expression by an IHC method (IHC3+), or (ii) biliary tract cancer given a score of 2+ for HER2 expression by an IHC method and determined as positive for HER2 amplificationby an ISH method (IHC2+ / ISH+). The ISH method may include fluorescencein situhybridization (FISH), dualin situhybridization (DISH), or silver-enhancedin situhybridization (SISH). In the present invention, a method for determining HER2 expression by immunohistochemistry (IHC) or a method for determining positivity or negativity to HER2 amplification by the ISH method may be used without particular limitation as long as it is recognized by those skilled in the art. For example, a determination method according to ASCO / CAP guidelines or NCCN guidelines may be used, without being limited thereto.

[0118] In the present invention, the biliary tract cancer may be HER2-expressing or -overexpressing biliary tract cancer. As used herein, the term "HER2-overexpressing biliary tract cancer" is not particularly limited as long as it is recognized as HER2-overexpressing cancer by those skilled in the art, but in a broad sense, the term may be interpreted to encompass all cases in which the HER2 expression level in the entire tumor is above a certain level. For example, the term may mean high levels of HER2 expression, a high percentage of HER2-expressing cells in the tumor, or expressing HER2 (HER2 expression). In another example, when the term "HER2-overexpressing biliary tract cancer" is interpreted narrowly, the term may be interpreted as expressing HER2 at high levels or having a high proportion of HER2-expressing cells in the tumor, without being limited thereto.

[0119] In the present invention, the HER2-expressing / overexpressing biliary tract cancer may be (i) biliary tract cancer given a score of 3+ for HER2 expression by an immunohistochemistry (IHC) method (IHC3+), (ii) biliary tract cancer given a score of 2+ for HER2 expression by an immunohistochemistry (IHC) method and determined as positive for HER2 amplification by an ISH method (IHC2+ / ISH+), or (iii) biliary tract cancer determined as positive for HER2 amplification by an ISH method (ISH+). The ISH method may include FISH (fluorescencein situhybridization), DISH (dualin situhybridization), or SISH (silver-enhancedin situhybridization). Any method for determining HER2 expression by immunohistochemistry (IHC) or determining positivity or negativity to HER2 amplification by the ISH method may be used without particular limitation as long as it is recognized by those skilled in the art. For example, a determination method according to ASCO / CAP guidelines or NCCN guidelines may be used, without being limited thereto.

[0120] As an example, the biliary tract cancer in the present invention may be IHC3+ biliary tract cancer or IHC2+ / ISH+ biliary tract cancer.

[0121] As an example, the biliary tract cancer in the present invention may be IHC3+ biliary tract cancer.

[0122] As an example, the biliary tract cancer in the present invention may be IHC2+ / ISH+ biliary tract cancer.

[0123] In addition, in the present invention, the HER2-expressing / overexpressing biliary tract cancer may be recognized as HER2-expressing / overexpressing cancer by a person skilled in the art through Southern blotting, a polymerase chain reaction (PCR) technique such as quantitative real-time PCR (qRT-PCR), or next-generation sequencing (NGS), without being limited thereto.

[0124] As an example, the HER2-expressing / overexpressing biliary tract cancer may mean that the proportion of HER2-expressing tumor cells among the total tumor cells constituting the biliary tract cancer is 50 to 99.95%, 50 to 99.5%, 50 to 99%, 50 to 95%, 50 to 90%, 60 to 99.95%, 60 to 99.5%, 60 to 99%, 60 to 95%, 60 to 90%, 70 to 99.95%, 70 to 99.5%, 70 to 99%, 70 to 95%, or 70 to 90%, based on the number of cells, the volume of cells, or the weight of cells.

[0125] Meanwhile, in the present invention, the biliary tract cancer may be HER2 low-expressing biliary tract cancer. As used herein, the term "HER2 low-expressing biliary tract cancer" is not particularly limited as long as it is recognized as HER2 low-expressing cancer by those skilled in the art, but in a broad sense, it may be interpreted to encompass all cases where the HER2 expression level in the entire tumor is below a certain level. For example, the term may mean a low expression level of HER2, a low percentage of HER2-expressing cells in the tumor, or no expression of HER2. In another example, when the term "HER2 low-expressing biliary tract cancer" is interpreted narrowly, it may be interpreted to mean a low expression level of HER2 or a low percentage of HER2-expressing cells in the tumor, without being limited thereto.

[0126] In the present invention, the "HER2 low-expressing biliary tract cancer" may be (i) biliary tract cancer given a score of 2+ for HER2 expression by immunohistochemistry (IHC) and determined as negative for HER2 amplification by an ISH method (IHC2+ / ISH-), or (ii) biliary tract cancer given a score of 1+ for HER2 expression by an immunohistochemistry method (IHC1+).

[0127] In addition, in the present invention, the HER2 low-expressing biliary tract cancer may be recognized as HER2 low-expressing cancer by a person skilled in the art through Southern blotting, a polymerase chain reaction (PCR) technique such as quantitative real-time PCR (qRT-PCR), or next-generation sequencing (NGS), without being limited thereto.

[0128] As an example, the HER2 low-expressing biliary tract cancer may mean that the proportion of HER2-expressing tumor cells among the total tumor cells constituting the biliary tract cancer is 0.05 to 50%, 0.5 to 50%, 1 to 50%, 5 to 50%, 10 to 50%, 0.05 to 40%, 0.5 to 40%, 1 to 40%, 5 to 40%, 10 to 40%, 0.05 to 30%, 0.5 to 30%, 1 to 30%, 5 to 30%, or 10 to 30%, based on the number of cells, the volume of cells, or the weight of cells.

[0129] In the present invention, the subject may include a subject having primary biliary tract cancer (where cancer cells are first formed) or secondary (metastatic) biliary tract cancer (biliary tract cancer formed by cancer cell spread from the primary cancer to bile duct tissue).

[0130] In the present invention, the subject may include a subject having metastatic biliary tract cancer.

[0131] In the present invention, the subject may include a subject having locally advanced biliary tract cancer. Here, "locally advanced biliary tract cancer" means a case including at least one of the following: no other organ metastasis; case where the biliary tract cancer has invaded the bilateral intrahepatic bile duct branches to a degree to which complete resection is impossible; case where the biliary tract cancer has invaded the bilateral hepatic artery or the common hepatic artery; case where the biliary tract cancer has invaded the portal vein to a degree to which reconstruction after resection is impossible; and case where findings suggesting lymph node metastasis are clear in tissue examination or imaging examination.

[0132] In the present invention, the subject may include a subject having advanced unresectable biliary tract cancer.

[0133] In the present invention, the subject may include a subject having pathologically or cytologically confirmed biliary tract cancer that is not a subject of curative resection, transplantation or resection therapy.

[0134] In the present invention, the subject may include a subject that has not received another therapy, for example, chemotherapy, hormonal therapy, targeted therapy, immunotherapy, other antitumor agents, or radiotherapy.

[0135] In the present invention, the subject may include a subject who has received first-line treatment as another therapy, for example, chemotherapy, hormonal therapy, targeted therapy, immunotherapy, other antitumor agents, or radiotherapy.

[0136] In the present invention, the subject may include a subject who has shown effective alleviation or treatment of biliary tract cancer in response to another therapy, for example, chemotherapy, hormonal therapy, targeted therapy, immunotherapy, other antitumor agents, or radiotherapy.

[0137] In the present invention, the subject may include a subject having biliary tract cancer that is refractory or intolerant to, or inadequately controlled by, another therapy, for example, chemotherapy, hormonal therapy, targeted therapy, immunotherapy, other antitumor agents, or radiotherapy. The term "refractory" as used herein means that the cancer is refractory to the therapy if the therapy does not significantly reduce the growth of the cancer, for example, if the tumor continues to grow or the size thereof does not decrease, or if the tumor starts to grow again after a period of response. The term "intolerant" means that the pharmacological action of the drug is abnormally increased, resulting in addiction symptoms at abnormally low doses. In addition, the term "inadequately controlled" includes, without limitation, cases where the therapy does not provide clinical benefit, such as cases where biliary tract cancer progresses despite the therapy, metastasis to other parts occurs, or disease progression is suppressed but then the disease relapses. Refractory to or inadequate control by therapy can be determined ex vivo by testing a sample (e.g., tumor biopsy sample) for cancer cell killing or growth inhibition, and / or in the clinical setting by observing (e.g., using an imaging technology, including MRI) that a patient treated with the therapy is not responding to treatment.

[0138] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to, or inadequately controlled by, standard therapy.

[0139] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to chemotherapy, targeted therapy or immunotherapy, or that is inadequately controlled by standard therapy.

[0140] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to chemotherapy, for example, but not limited to, therapy with gemcitabine, cisplatin, paclitaxel, 5-fluorouracil, leucovorin, oxaliplatin, capecitabine, irinotecan, abraxane, carboplatin, pemetrexed, or a combination of two or more thereof, or the targeted or immunotherapeutic agents targeting HER2, PD-1, PD-L1, EGFR, VEGFR, CTLA-4, etc., or is inadequately controlled by standard therapy. Here, non-limiting examples of the targeted or immunotherapeutic agents include, but are not limited to, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, trastuzumab rezetecan, zanidatamab (ZW25), zanidatamab zovodotin (ZW49), pertuzumab, lapatinib, tucatinib, margetuximab, disitamab vedotin (RC48-ADC), MRG002 ADC, durvalumab, nivolumab, pembrolizumab, bevacizumab, cetuximab, lenvatinib, ramucirumab, SAR443216, BDC-1001, atezolizumab, or ipilimumab.

[0141] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to, or by inadequately controlled by, gemcitabine, cisplatin, paclitaxel, 5-fluorouracil, leucovorin, oxaliplatin, capecitabine, irinotecan, abraxane, carboplatin, pemetrexed, pembrolizumab, bevacizumab, cetuximab, lenvatinib, ramucirumab, or a combination of two or more thereof.

[0142] As an example, the subject in the present invention may include a subject to whom gemcitabine, cisplatin, paclitaxel, 5-fluorouracil, leucovorin, oxaliplatin, capecitabine, irinotecan, abraxane, carboplatin, pemetrexed, pembrolizumab, bevacizumab, cetuximab, lenvatinib, ramucirumab, or a combination of two or more thereof has been administered, but who has biliary tract cancer that is refractory or intolerant to, or inadequately controlled by, such a therapeutic agent.

[0143] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to, or inadequately controlled by, gemcitabine or cisplatin.

[0144] As an example, the subject in the present invention may include a subject to whom gemcitabine or cisplatin has been administered, but who has biliary tract cancer that is refractory to, or inadequately controlled by, the therapeutic agent.

[0145] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to anti-HER2 therapy targeting HER2, or that is inadequately controlled by the therapy.

[0146] As an example, the subject in the present invention may include a subject to whom an anti-HER2 therapy targeting HER2 has been administered, but who has biliary tract cancer that is refractory to, or inadequately controlled, by the therapy.

[0147] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to, or inadequately controlled by, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, trastuzumab rezetecan, zanidatamab (ZW25), zanidatamab zovodotin (ZW49), pertuzumab, tucatinib, lapatinib, SAR443216, BDC-1001, or a combination of two or more thereof as anti-HER2 therapy.

[0148] As an example, the subject in the present invention may include a subject to whom trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, trastuzumab rezetecan, zanidatamab (ZW25), zanidatamab zovodotin (ZW49), pertuzumab, tucatinib, lapatinib, SAR443216, BDC-1001, or a combination of two or more thereof as anti-HER2 therapy has been administered, but who has biliary tract cancer that is refractory to, or inadequately controlled by, the therapeutic agent.

[0149] As an example, the subject in the present invention may include a subject having biliary tract cancer that is refractory or intolerant to, or inadequately controlled by, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, or trastuzumab rezetecan as anti-HER2 therapy.

[0150] As an example, the subject in the present invention may include a subject to whom trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, or trastuzumab rezetecan has been administered, but who has biliary tract cancer that is refractory to, or inadequately controlled by, such a therapeutic agent.

[0151] In the present invention, the subject may include a subject who has shown effective alleviation or treatment of biliary tract cancer disease in response to another therapy, for example, chemotherapy, hormonal therapy, targeted therapy, immunotherapy, other anti-tumor agents, or radiotherapy, but who subsequently has a relapsed or metastatic solid tumor.

[0152] The present invention, the subject may include a subject with a solid tumor who has been treated with another therapy, such as chemotherapy, hormonal therapy, targeted therapy, immunotherapy, other anti-tumor agents, or radiotherapy, but in whom biliary tract cancer that is resistant to the therapy subsequently relapsed.

[0153] In the present invention, the subject may include a subject having biliary tract cancer for whom conventional therapy, such as anticancer chemotherapy, targeted therapy, or immunotherapy for biliary tract cancer or other cancer patients, cannot be recommended due to its toxic side effects.

[0154] In the present invention, the subject may include a subject whose cumulative administration dose of doxorubicin is 360 mg / m2(body surface area) or less at the time of initiation of administration of the antibody of the present invention.

[0155] In the present invention, the subject may include a subject whose cumulative administration dose of epirubicin is 720 mg / m2(body surface area) or less at the time of initiation of administration of the antibody of the present invention.

[0156] Dosage and Regimen

[0157] In the present invention, the amount of the anti-HER2 / anti-4-1BB bispecific antibody administered to a subject is, generally, a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means an amount of the anti-HER2 / anti-4-1BB bispecific antibody that results in the following: (a) a reduction in the severity or duration of symptoms or signs of biliary tract cancer; (b) inhibition of the growth of biliary tract cancer, or an increase in tumor necrosis, tumor shrinkage, and / or tumor disappearance; 　(c) delay in the growth and development of biliary tract cancer; (d) inhibition of biliary tract cancer metastasis; (e) prevention of recurrence of biliary tract cancer growth; (f) an increase in survival of a subject with biliary tract cancer; and / or (g) a reduction in the use or need for conventional anticancer therapy (e.g., reduced or eliminated use of chemotherapeutic or cytotoxic agents) as compared to an untreated subject.

[0158] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 10 mg to about 5000 mg, about 10 mg to about 4000 mg, about 10 mg to about 3000 mg, about 10 mg to about 2100 mg, about 10 mg to about 1500 mg, about 20 mg to about 5000 mg, about 20 mg to about 4000 mg, about 20 mg to about 3000 mg, about 20 mg to about 2100 mg, about 20 mg to about 1500 mg, about 50 mg to about 5000 mg, about 50 mg to about 4000 mg, about 50 mg to 3000 mg, about 50 mg to about 2100 mg, about 50 mg to about 1500 mg, about 75 mg to about 2100 mg, about 75 mg to about 1500 mg, about 100 mg to about 3000 mg, about 100 mg to about 2100 mg, about 100 mg to about 1500 mg, or about 100 mg to about 1300 mg.

[0159] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2500 mg, about 3000 mg, about 3500 mg, about 4000 mg, or about 5000 mg, without being limited thereto.

[0160] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 0.1 mg / kg or more, about 0.3 mg / kg or more, about 0.5 mg / kg or more, about 0.75 mg / kg or more, about 1 mg / kg or more, about 1.5 mg / kg or more, about 2 mg / kg or more, about 3 mg / kg or more, about 5 mg / kg or more, about 6 mg / kg or more, about 8 mg / kg or more, about 10 mg / kg or more, or about 12 mg / kg or more, without being limited thereto.

[0161] In addition, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 50 mg / kg or less, about 40 mg / kg or less, about 30 mg / kg or less, about 25 mg / kg or less, about 20 mg / kg or less, about 15 mg / kg or less, about 12 mg / kg or less, about 10 mg / kg or less, about 8 mg / kg or less, or about 6 mg / kg or less, without being limited thereto.

[0162] In addition, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 0.1 mg / kg or more, about 0.3 mg / kg or more, about 0.5 mg / kg or more, about 0.75 mg / kg or more, about 1 mg / kg or more, about 1.5 mg / kg or more, about 2 mg / kg or more, about 3 mg / kg or more, about 5 mg / kg or more, about 6 mg / kg or more, about 8 mg / kg or more, about 10 mg / kg or more, or about 12 mg / kg or more, and about 50 mg / kg or less, about 40 mg / kg or less, about 30 mg / kg or less, about 25 mg / kg or less, about 20 mg / kg or less, about 15 mg / kg or less, about 12 mg / kg or less, about 10 mg / kg or less, about 8 mg / kg or less, or about 6 mg / kg or less, without being limited thereto.

[0163] In addition, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.3 mg / kg to about 50 mg / kg, about 0.3 mg / kg to about 40 mg / kg, about 0.3 mg / kg to about 30 mg / kg, about 0.75 mg / kg to about 30 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 25 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 15 mg / kg, about 1 mg / kg to about 12 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 8 mg / kg, about 1 mg / kg to about 6 mg / kg, about 1.5 mg / kg to about 30 mg / kg, about 1.5 mg / kg to about 25 mg / kg, about 1.5 mg / kg to about 20 mg / kg, about 1.5 mg / kg to about 15 mg / kg, about 1.5 mg / kg to about 12 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 8 mg / kg, about 1.5 mg / kg to about 6 mg / kg, about 2 mg / kg to about 30 mg / kg, about 2 mg / kg to about 25 mg / kg, about 2 mg / kg to about 20 mg / kg, about 2 mg / kg to about 15 mg / kg, about 2 mg / kg to about 12 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 8 mg / kg, about 2 mg / kg to about 6 mg / kg, about 3 mg / kg to about 30 mg / kg, about 3 mg / kg to about 25 mg / kg, about 3 mg / kg to about 20 mg / kg, about 3 mg / kg to about 15 mg / kg, about 3 mg / kg to about 12 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3 mg / kg to about 8 mg / kg, about 3 mg / kg to about 6 mg / kg, about 5 mg / kg to about 30 mg / kg, about 5 mg / kg to about 25 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 15 mg / kg, about 5 mg / kg to about 12 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 8 mg / kg, about 5 mg / kg to about 6 mg / kg, about 6 mg / kg to about 30 mg / kg, about 6 mg / kg to about 25 mg / kg, about 6 mg / kg to about 20 mg / kg, about 6 mg / kg to about 15 mg / kg, about 6 mg / kg to about 12 mg / kg, about 6 mg / kg to about 10 mg / kg, or about 6 mg / kg to about 8 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 25 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 15 mg / kg, or about 10 mg / kg to about 12 mg / kg, without being limited thereto.

[0164] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject at a dose of about 0.3 mg / kg, about 0.75 mg / kg, about 1.5 mg / kg, about 3 mg / kg, about 6 mg / kg, about 12 mg / kg, about 20 mg / kg, or about 30 mg / kg.

[0165] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 0.3 mg / kg, without being limited thereto.

[0166] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 0.75 mg / kg, without being limited thereto.

[0167] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 1.5 mg / kg, without being limited thereto.

[0168] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 3 mg / kg, without being limited thereto.

[0169] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 6 mg / kg, without being limited thereto.

[0170] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 12 mg / kg, without being limited thereto.

[0171] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 20 mg / kg, without being limited thereto.

[0172] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject in a therapeutically effective amount of about 30 mg / kg, without being limited thereto.

[0173] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject at a dosing frequency of about twice a week, about once a week, about once every 10 days, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once a month, about once every six weeks, about once every seven weeks, about once every eight weeks, or about once every two months.

[0174] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject at a frequency of about once every three weeks, without being limited thereto.

[0175] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 0.3 mg / kg.

[0176] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 0.75 mg / kg.

[0177] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 1.5 mg / kg.

[0178] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 3 mg / kg.

[0179] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 6 mg / kg.

[0180] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 12 mg / kg.

[0181] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 20 mg / kg.

[0182] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject about once every three weeks at a dose of about 30 mg / kg.

[0183] In the present invention, the route of administration of the anti-HER2 / anti-4-1BB bispecific antibody may include, but is not limited to, oral administration or parenteral administration. Here, parenteral administration may include intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration, but is preferably intravenous administration.

[0184] In the present invention, when the anti-HER2 / anti-4-1BB bispecific antibody is administered intravenously to a subject, the anti-HER2 / anti-4-1BB bispecific antibody or the pharmaceutical composition containing the same may be administered by infusion over a period of about 10 minutes to about 5 hours, about 10 minutes to about 3 hours, about 20 minutes to about 3 hours, about 30 minutes to about 3 hours, about 10 minutes to about 2 hours, about 20 minutes to about 2 hours, about 30 minutes to about 2 hours, about 10 minutes to about 90 minutes, about 20 minutes to about 90 minutes, or about 30 minutes to about 90 minutes.

[0185] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered intravenously to a subject over about 30 minutes.

[0186] As an example, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered intravenously to a subject over about 90 minutes.

[0187] In the present invention, the anti-HER2 / anti-4-1BB bispecific antibody may be administered to a subject for at least 3 weeks, at least 6 weeks, at least 9 weeks, at least 11 weeks, at least 12 weeks, at least 15 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 21 weeks, or at least 24 weeks, without being limited thereto. Here, the administration period may mean the minimum period required to achieve a therapeutically meaningful effect from the time of the first administration of the antibody to the subject. The antibody may be administered at least once during the administration period.

[0188] In the present invention, the anti-HER2 / anti-4-1BB bispecific antibody may be administered to a subject at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, or at least eight times, without being limited thereto. Here, the number of administrations may mean the minimum number of administrations required to achieve a therapeutically meaningful effect after the first administration of the antibody to the subject. The antibody may be administered at least once during the administration period.

[0189] The anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject as a first-line treatment. As used herein, the term "first-line treatment" refers to a treatment that is generally accepted as the first treatment for a patient, taking into account the type and stage of the cancer.

[0190] Additionally, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered to a subject as a second-line treatment. As used herein, the term "second-line treatment" refers to a treatment that is typically administered when first-line treatment does not offer the desired efficacy.

[0191] The anti-HER2 / anti-4-1BB bispecific antibodies of the present invention may be administered to a subject alone or in combination with other types of therapy (e.g., radiotherapy, chemotherapy, hormonal therapy, immunotherapy, and antitumor agents).

[0192] In particular, the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may be administered in combination with an additional anticancer therapeutic agent. Here, additional anticancer therapeutic agents that may be used include anticancer chemotherapeutic agents, hormonal therapeutic agents, targeted therapeutic agents or immunotherapeutic agents. Non-limiting examples of the anticancer chemotherapeutic agents include gemcitabine, cisplatin, paclitaxel, 5-fluorouracil, leucovorin, oxaliplatin, capecitabine, irinotecan, abraxane, carboplatin, pemetrexed, or combinations thereof, and non-limiting examples of the targeted or immunotherapeutic agents include trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, trastuzumab rezetecan, zanidatamab (ZW25), zanidatamab zovodotin (ZW49), pertuzumab, lapatinib, tucatinib, margetuximab, disitamab vedotin (RC48-ADC), MRG002 ADC, durvalumab, nivolumab, pembrolizumab, atezolizumab, ipilimumab, bevacizumab, cetuximab, lenvatinib, ramucirumab, SAR443216, BDC-1001, or combinations thereof. However, additional anticancer therapeutic agents that may be used in combination with the anti-HER2 / anti-4-1BB bispecific antibody of the present invention may include not only drugs listed as drugs for treating biliary tract cancer in Simile et al. (2019) Medicina 55: 42, but also drugs used as drugs for treating biliary tract cancer in the art, or drugs currently undergoing clinical trials for the treatment of biliary tract cancer but not yet approved by the FDA.

[0193] In the present invention, when the anti-HER2 / anti-4-1BB bispecific antibody is used in combination with another type of therapeutic agent, they may be administered simultaneously or sequentially.

[0194] Results of Prevention, Alleviation or Treatment of Biliary Tract Cancer

[0195] In the present invention, when the anti-HER2 / anti-4-1BB bispecific antibody is administered to the above-described subject using the method according to the present invention, biliary tract cancer can be treated safely and effectively.

[0196] As used herein, the term "alleviation or treatment of biliary tract cancer" means alleviating or eliminating at least one of the symptoms or signs of biliary tract cancer, temporarily or permanently eliminating the cause of the symptoms, delaying or inhibiting tumor growth, reducing the tumor cell load or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis or disappearance, preventing or inhibiting tumor recurrence, inhibiting tumor invasion, preventing or inhibiting tumor metastasis, inhibiting recurrent tumor growth, inhibiting metastatic tumor growth, or increasing the duration of survival of the subject. In the present invention, the effect on the prevention, alleviation, or treatment of the biliary tract cancer described above can be determined by measuring lesions on chest X-ray or CT or MRI films, or by evaluating responsiveness to therapy using cytology or histology.

[0197] As an example, when the anti-HER2 / anti-4-1BB bispecific antibody of the present invention is administered, the size of the biliary tract cancer in the subject may be reduced. The size of the biliary tract cancer may be reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size before administration, without being limited thereto.

[0198] The subject administered the anti-HER2 / anti-4-1BB bispecific antibody according to the present invention may exhibit a complete response (CR), a partial response (PR), stable disease (SD), immune-related complete response (irCR), immune-related partial response (irPR), or immune-related stable disease (irSD).

[0199] With respect to the terms "complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD)" as used herein, as described in the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) (Eisenhauer et al., 2009), "complete response (CR)" refers to disappearance of all target lesions, "partial response (PR)" refers to at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters, and "progressive disease (PD)" refers to at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. Additionally, "stable disease (SD)" refers to neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study. Meanwhile, "duration of response (DoR)" can be defined as the time from the date of the first documented response (CR or PR) to the date of documented progression or death after response.

[0200] The subject administered the anti-HER2 / anti-4-1BB bispecific antibody according to the present invention may exhibit progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the initial administration.

[0201] Kit or Article of Manufacture

[0202] The present invention provides a kit or article of manufacture comprising the anti-HER2 / anti-4-1BB bispecific antibody.

[0203] The kit or article of manufacture according to the present invention may further comprise instructions for use of the anti-HER2 / anti-4-1BB bispecific antibody according to the present invention. The instructions include instructions for use in treating biliary tract cancer by administering the anti-HER2 / anti-4-1BB bispecific antibody according to the present invention to a subject.

[0204] In addition, the kit or article of manufacture according to the present invention may further comprise a container. Suitable containers include, for example, but not limited to, bottles, vials (e.g., dual chamber vials), syringes (e.g., single or dual chamber syringes), and test tubes.

[0205] As an example, the kit or article of manufacture according to the present invention may comprise a vial container.

[0206] Additionally, the kit or article of manufacture according to the present invention may further comprise a label or package insert on or associated with the container, which may indicate directions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous (e.g., intravenous infusion), or other modes of administration for treating biliary tract cancer as described herein in a subject. The container holding the formulation may be a single-use vial or a multi-use vial, which allows for repeat administrations of the reconstituted formulation.　The article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0207] In the kit or article of manufacture according to the present invention, the anti-HER2 / anti-4-1BB bispecific antibody according to the present invention may be present in a container as a lyophilized powder. Here, the lyophilized powder may be in a hermetically sealed container, such as a vial, an ampoule or sachette, indicating the quantity of the active agent. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline may be, for example, provided, optionally as part of the kit, so that the ingredients may be mixed prior to administration. In the kit or article of manufacture according to the present invention, the anti-HER2 / anti-4-1BB bispecific antibody according to the present invention may be present in a container as a liquid pharmaceutical composition further comprising a pharmaceutically acceptable carrier, lubricant, diluent, excipient, stabilizer, or the like.

[0208] The present invention provides a pharmaceutical composition and a therapeutic method that can exhibit an effective antitumor effect against cancer, particularly biliary tract cancer, and excellent safety, by administering a specific anti-HER2 / anti-4-1BB bispecific antibody.

[0209] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

[0210] Examples

[0211] [Preparation Example 1] Preparation of Anti-HER2 / Anti-4-1BB Bispecific Antibody

[0212] For the study below, a bispecific antibody capable of binding to both HER2 and 4-1BB was prepared and named "bispecific antibody AHYM12". The bispecific antibody is composed of: the heavy chain of trastuzumab (Genentech; DrugBank Accession No. DB00072; human IgG1 Kappa monoclonal antibody) consisting of the amino acid sequence shown in Table 10 below; an anti-4-1BB scFv having the structure (N')-VL-linker-VH-(C'); and the light chain of trastuzumab. Here, the heavy component (SEQ ID NO: 29) was configured such that the 4-1BB scFv fragment was linked to the C-terminus of anti-HER2 IgG by an 18-amino acid peptide linker consisting of (GS)9(SEQ ID NO: 33). The method for preparing this bispecific antibody AHYM12 is described in detail in International Patent Publication WO 2021-020846.

[0213] ClassificationAmino acid sequence information (N-terminus → C-terminus)Heavy component (①+②+③)① Heavy chain of anti-HER2 antibodyEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27)② Peptide linkerGSGSGSGSGSGSGSGSGS (SEQ ID NO: 33)③Anti-4-1BB scFvVLQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL (SEQ ID NO: 17)Peptide linkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32)VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS (SEQ ID NO: 16)Light componentLight chain of anti-HER2 antibodyDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28)

[0214] [Example 1] Phase 1 / 2 Clinical Trial of Bispecific Antibody AHYM12 on Patients with HER2-Positive Locally Advanced or Metastatic Cancer

[0215] A phase 1 / 2 clinical trial as a first-in-human study is being conducted to evaluate the safety, tolerability, pharmacokinetics and antitumor activity of the bispecific antibody AHYM12 of Preparation Example 1 as monotherapy in patients with HER2-positive locally advanced or metastatic cancer.

[0216] Part 1 of the study was a dose-escalation phase, in which the dose was escalated from a starting dose of 0.3 mg / kg to a total of eight dose levels (0.3, 0.75, 1.5, 3, 6, 12, 20, and 30 mg / kg) using a modified accelerated titration design (ATD) and the Bayesian optimal interval design (BOIN) to determine the maximum tolerated dose (MTD) of the bispecific antibody AHYM12 in patients with HER2-positive locally advanced or metastatic cancer that is refractory or intolerant to standard therapy or for which there is no available standard therapy. After the last patient in each dose cohort completed Cycle 1 (i.e., 21 days from first dose), the Safety Review Committee (SRC) determined whether to escalate to the next dose level based on safety and available data. By analyzing the data collected in Part 1, two candidate doses for selection of the MTD and / or recommended phase 2 dose (RP2D) were determined, and the dose expansion phase (Part 2) was initiated at these doses.

[0217] Part 2 of the study, a dose expansion phase, includes two cohorts:

[0218] Cohort 1: Patients with HER2-positive (IHC3+ or IHC2+ / ISH+) locally advanced / unresectable or metastatic biliary tract cancer (including intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, gallbladder cancer, and ampullary adenocarcinoma) who have failed or are refractory to at least one prior therapy based on gemcitabine and / or cisplatin with or without an immune checkpoint inhibitor according to local standard of care; and

[0219] Cohort 2: Patients with HER2-positive (IHC3+ or IHC2+ / ISH+) locally advanced / unresectable or metastatic solid cancer (excluding breast cancer, gastric or gastroesophageal junction adenocarcinoma, and biliary tract cancer) who have received all available standard therapies known to provide clinical benefit and who experienced confirmed disease progression or have no other available treatment options due to intolerance.

[0220] Cohort 1 includes two RP2D candidate dose groups, and a total of 50 patients are randomly assigned to each candidate dose group at a 1:1 ratio (i.e., 25 patients per candidate dose group). Once the RP2D is determined, an additional 15 patients are assigned to the determined RP2D dose group. Cohort 2 enrolls 40 patients, and the safety, tolerability, and efficacy of the bispecific antibody AHYM12 are evaluated at the RP2D.

[0221] Objectives

[0222] The primary objective of Part 1 is to evaluate the safety and tolerability of the bispecific antibody AHYM12, with secondary objectives to characterize PK, explore immunogenicity, and evaluate antitumor efficacy as assessed by the investigator according to RECIST v1.1. The evaluation of overall survival of bispecific antibody AHYM12-administerd patients, the evaluation of immune-related efficacy as assessed by the investigator according to iRECIST (RECIST 1.1 modified for immune-based therapies), and the evaluation of biomarkers associated with the bispecific antibody AHYM12 are exploratory.

[0223] The primary objective of Part 2 is to evaluate the objective response rate of the bispecific antibody AHYM12 at RP2D as assessed by a blinded independent central review (BICR) according to RECIST v1.1, with secondary objectives to evaluate the safety and tolerability of the bispecific antibody AHYM12 at RP2D, characterize PK, explore immunogenicity, evaluate antitumor efficacy as evaluated by BICR according to RECIST v1.1, and evaluate overall survival of bispecific antibody AHYM12-administered patients. Evaluation of immune-related efficacy as evaluated by the investigator according to iRECIST, evaluation of biomarkers associated with the bispecific antibody AHYM12, exploration of the feasibility of artificial intelligence (AI)-based tissue analysis, and evaluation of the impact of the bispecific antibody AHYM12 on patients' disease-related symptoms and health-related quality of life are exploratory.

[0224] Patients

[0225] Adult male or female patients aged ≥18 years with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and a life expectancy of at least 3 months in the opinion of the clinical investigator are eligible to participate in the trial. Female patients of childbearing potential must have a negative pregnancy test result, and both the man and the woman must agree to use contraception during the study period. In addition, the previous cumulative doxorubicin dose must be ≤360 mg / m2and the previous cumulative epirubicin dose must be ≤720 mg / m2.

[0226] In Part 1, included were patients with pathologically confirmed HER2-positive (IHC3, or IHC2+ / ISH+) locally advanced or metastatic solid cancer who were refractory or intolerant to standard therapy, or experienced disease progression on standard therapy, or for whom there is no available standard therapy. In particular, patients with breast cancer or gastric cancer (or gastroesophageal junction adenocarcinoma) met the following criteria: Patients with breast cancer who were locally assessed as HER2-positive and have received ≥3 prior lines of therapy including at least one trastuzumab-based therapy in the palliative care setting (if the breast cancer recurred within 12 months after adjuvant trastuzumab-based therapy, the adjuvant trastuzumab-based therapy could be considered first-line trastuzumab therapy); and patients with gastric cancer who were locally assessed as HER2-positive and have received ≥2 prior lines of therapy including at least one trastuzumab-based therapy in the palliative care setting.

[0227] In Part 2, included were patients with HER2-positive (IHC3+, or IHC2+ / ISH+) locally advanced / unresectable or metastatic biliary tract cancer (including intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, gallbladder cancer, and ampullary adenocarcinoma) who have failed or are refractory to at least one prior therapy based on gemcitabine and / or cisplatin with or without an immune checkpoint inhibitor according to current standard of care (Cohort 1), and patients with HER2-positive (IHC3+, or IHC2+ / ISH+) locally advanced / unresectable or metastatic solid cancer other than breast cancer, gastric or gastroesophageal junction adenocarcinoma, and biliary tract cancer who have received all available standard of care known to provide clinical benefit and who experienced confirmed disease progression or have no remaining available treatment options due to intolerance (Cohort 2).

[0228] Patients were excluded from the study if one or more of the following criteria applied:

[0229] 1. Known uncontrolled central nervous system (CNS) metastases, spinal cord compression, and / or malignant meningitis

[0230] 2. History of acute coronary syndrome, including myocardial infarction, coronary artery bypass grafting, unstable angina, coronary angioplasty, or stent placement, within the past 24 weeks.

[0231] 3. Past or current history of heart failure grade 2, 3, or 4 as defined by the New York Heart Association (NYHA) functional classification system.

[0232] 4. History of ejection fraction falling below the lower limit of normal during administration of trastuzumab and / or pertuzumab

[0233] 5. Medical, psychiatric, cognitive, or other conditions that may impair the patient's ability to understand patient information, give written informed consent, comply with the clinical trial protocol, or complete the trial.

[0234] 6. Any serious concomitant disease or condition (including active infection or cardiac arrhythmia) that, in the investigator's option, makes the patient inappropriate for study participation.

[0235] 7. History of (noninfectious) interstitial lung disease (ILD) or pneumonia requiring steroids, or evidence of current ILD or pneumonia.

[0236] 8. History of second primary cancer, excluding curatively treated nonmelanoma skin cancer, curatively treated intraepithelial carcinoma of the cervix or breast, or any other malignancy with no known active disease and no treatment administered in the past 2 years.

[0237] 9. Human immunodeficiency virus (HIV) infection or active chronic hepatitis B or C.

[0238] 10. History of infusion reactions to any component / excipient of the bispecific antibody AHYM12

[0239] 11. History of severe allergic or anaphylactic reactions to antibodies or infused therapeutic proteins

[0240] 12. Systemic steroid therapy (>10 mg daily prednisone or equivalent steroids) or any other form of immunosuppressive therapy within 7 days prior to the first dose of test treatment (Note: topical, inhaled, nasal and ophthalmic steroids are not prohibited);

[0241] 13. Autoimmune disease that has required systemic treatment (i.e., with use of disease modifying agents, corticosteroids, or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or the like) is allowed for patients with controlled autoimmune diseases.

[0242] 14. Patients who have not recovered from the adverse effect of previous anticancer treatments to pretreatment baseline or Grade 1 except for alopecia, anemia (hemoglobin levels must meet the study inclusion criteria) and peripheral neuropathy (which must have recovered to ≤ Grade 2), nausea and diarrhea during treatment

[0243] 15. Those who received clinical trial treatment within 3 weeks prior to the first dose of bispecific antibody AHYM12 treatment

[0244] 16. Those who received cytotoxic chemotherapy within 3 weeks (6 weeks for nitrosourea and mitomycin C) prior to the first dose of bispecific antibody AHYM12 treatment

[0245] 17. Those who received radiation therapy within 2 weeks of the first dose of the bispecific antibody AHYM12. However, limited radiotherapy to non-visceral sites, such as metastases in the arms, legs, or bones, is excluded;

[0246] 18. Those who received treatment with immunotherapy, biological therapy, targeted small molecules, or hormonal therapy within 3 weeks prior to the first dose of bispecific antibody AHYM12 treatment

[0247] 19. Those who received trastuzumab or ado-trastuzumab emtansine or other developmental drug that engages the same epitope as trastuzumab within 4 weeks prior to the first dose of bispecific antibody AHYM12 treatment

[0248] 20. Concurrent enrollment in another therapeutic clinical trial;

[0249] 21. Major surgery within 3 weeks prior to the first dose of bispecific antibody AHYM12 treatment

[0250] 22. Those who received a live vaccine within 4 weeks prior to the first dose of bispecific antibody AHYM12 treatment and plan to receive a live vaccine during the study

[0251] 23. Previous treatment with 4-1BB / CD137 modulatory agents

[0252] 24. Inadequate organ function defined as follows:

[0253] 1) Serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 3 X upper limit normal (ULN)

[0254] 2) Total serum bilirubin > 1.5 X ULN

[0255] 3) Part 1: Serum creatinine > 1.5 X ULN or glomerular filtration rate calculated by the Cockcroft-Gault formula < 50 mL / min; Part 2: Glomerular filtration rate calculated by the Cockcroft-Gault formula < 30 mL / min

[0256] 4) Hemoglobin < 9g / dL

[0257] 5) Absolute neutrophil count < 1500 / mm3

[0258] 6) Lymphocyte count < 800 cells / μL

[0259] 7) Platelet count < 75,000 / mm3

[0260] 8) Fridericia-corrected QT interval (QTcF) prolongation > 480 ms based on triplicate 12-lead electrocardiograms

[0261] 9) Left ventricular ejection fraction (LVEF) <50% by echocardiography (ECHO) or multiple gated acquisition (MUGA)

[0262] Treatment

[0263] As a single agent (monotherapy), the bispecific antibody AHYM12 was administered as an intravenous (IV) solution. Patients received the bispecific antibody AHYM12 once every 3 weeks (Q3W), and a cycle is defined as the time from the first day of administration to the first day of the next administration. If treatment is not delayed, one cycle is 3 weeks (1 cycle is 21 days). The bispecific antibody AHYM12 is administered as an intravenous infusion to each patient over 90 minutes on Day 1 of Cycle 1 (C1D1). If there is no infusion-related reaction after the first dose, the next dose is administered as an intravenous infusion to each patient over approximately 30 minutes. Regarding the infusion rate, if an infusion-related reaction occurs but recovers before the end of the infusion, it is considered that no infusion-related reaction occurred after the first dose. If symptoms of an infusion reaction are present, the infusion rate may be reduced or the infusion may be temporarily stopped, and symptomatic treatment may be administered.

[0264] The patient's body weight prior to administration on C1D1 is used to calculate the initial dose. If the patient's body weight increases or decreases by ≥10% of the body weight used to calculate the last dose, calculation is performed again using the actual body weight at that visit.

[0265] For patients who are unable to adhere to the dosing schedule according to the clinical trial protocol due to drug-related toxicity, temporary interruption of dosing and / or a reduction in the dose to a lower level is recommended to allow the patient to continue treatment. If dosing to the patient must be interrupted for a period exceeding 4 weeks from the scheduled dosing date, the patient may discontinue treatment.

[0266] Dose

[0267] In Part 1, the dose levels for dose escalation are 0.3, 0.75, 1.5, 3, 6, 12, 20, and 30 mg / kg, administered once every 3 weeks (Q3W).

[0268] The dose levels for Part 2 are the two doses for selection of MTD or RP2D determined in Part 1. MTD is selected based on isotonic regression after completion of Part 1 (Liu, S., & Yuan, Y. Bayesian optimal interval designs for phase I clinical trials. Journal of the Royal Statistical Society: Series C: Applied Statistics, 507-523.2015). Specifically, the dose whose isotonic estimate of the toxicity incidence rate is closest to the target toxicity incidence rate (25%) is selected as the MTD. If there are ties, the higher dose level will be selected when the isotonic estimate is lower than the target toxicity rate, and the lower dose level will be selected when the isotonic estimate is greater than or equal to the target toxicity rate. The two doses for selecting the RP2D are any other doses that do not exceed the MTD.

[0269] Efficacy Evaluation

[0270] Tumor assessments were performed using CT or MRI scans through the same tumor imaging as that at screening, every 6 weeks (±7 days) for the first 24 weeks after Day 1 of Cycle 1, and every 12 weeks (±7 days) thereafter until disease progression or before the start of subsequent anticancer therapy, regardless of dose delay.

[0271] Measurable or non-measurable target lesions and nontarget lesions and objective tumor response criteria were based on RECIST v1.1 guidelines (Eur J Cancer. 2009 Jan;45(2):228-47). Tumor response was assessed by the investigator as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), and in Part 2, assessment by an independent central imaging organization conducted a review alongside the investigator's tumor assessment.

[0272] The investigator also assessed the overall response rate according to iRECIST along with RECIST v1.1 (Lancet Oncol. 2017;18:e143-e152). Immunotherapy can cause immune cell infiltration, which can temporarily increase the size of cancer lesions or make previously undetectable lesions detectable. iRECIST is similar in many respects to RECIST v1.1 criteria, but has been modified to account for cases where increasing tumor burden or the appearance of new lesions does not reflect true tumor progression.

[0273] Objective response rate (ORR) is defined as the percentage of patients with at least one confirmed CR or PR before disease progression or relapse among patients having at least one measurable lesion at baseline.

[0274] Disease control rate (DCR) is defined as the proportion of patients with a best overall response (BOR) of CR, PR, or SD.

[0275] Duration of response (DoR) is defined as the time from the date of the first documented response for a confirmed response to the earlier of the dates of documented progression or death.

[0276] Progression-free survival (PFS) is defined as the time from the first dose of the bispecific antibody AHYM12 to the date of the earlier of objective disease progression based on RECIST v1.1 or death (from any cause in the absence of progression).

[0277] Exploratory Evaluation

[0278] Tumor tissue samples and blood samples are collected for exploratory biomarker analysis. Tumor tissue samples are used to analyze HER2, CD4, CD8, CD56, Perforin, FoxP3, and PD-L1 expression, and blood samples are used to analyze cytokines, soluble 4-1BB, and immunophenotypes (CD3, CD4, CD8, CD14, CD16, CD25, CD45RA, CD56, CD127, CD137, CD197, FoxP3, Ki67, Granzyme B, and PD-1).

[0279] Adverse Event

[0280] An adverse event (AE) is defined as any undesirable and unintended sign (including laboratory abnormalities), symptom, or disease that occurs in a patient and does not necessarily have a causal relationship with the investigational product (IP). AEs are evaluated according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v5.0.

[0281] Part 1 Test Results:

[0282] As of May 7, 2024, 32 patients across all indications were enrolled in Part 1 and treated with the single-agent bispecific antibody AHYM12. In demographic information at the time of data cutoff on May 24, 2024, the mean age of the patients was approximately 60 years, there were 20 males and 12 females, and the majority of the patients were Asian (n = 29, 90.6%), with no Hispanic or Latino patients. The tumor types of the enrolled patients included 14 (43.8%) biliary tract cancers, 9 (28.1%) gastric cancer or gastroesophageal junction adenocarcinomas, 3 (9.4%) salivary gland cancers, 2 (6.3%) breast cancers, 2 (6.3%) non-small cell lung cancers, 1 (3.1%) colorectal cancer, and 1 (3.1%) vaginal cancer. The median time since the first cancer diagnosis in the enrolled patients was approximately 29 months (range: approximately 6.9 to 111.6 months), and the patients all had stage IV cancer. As a result of performing HER2 testing, 24 out of the 32 patients were interpreted as HER2 IHC3+, and 8 patients were interpreted as HER2 IHC2+ / ISH+. Among all enrolled patients, 1 patient had brain metastasis.

[0283] The enrolled patients had received prior systemic therapy with gemcitabine, cisplatin, paclitaxel, fluorouracil, leucovorin, oxaliplatin, capecitabine, irinotecan, pembrolizumab, abraxane, bevacizumab, carboplatin, cetuximab, lenvatinib, pemetrexed, ramucirumab, or a combination of two or more thereof. Additionally, 17 of the 32 enrolled patients had previously received trastuzumab-based therapies (trastuzumab, trastuzumab emtansine, trastuzumab deruxtecan, or trastuzumab rezetecan) (Table 11 below), and 4 of the 14 patients with biliary tract cancer had previously received trastuzumab-based therapies.

[0284] Previous anticancer treatment in patients enrolled in Part 1Number of systemic therapiesNumber of patients01110283447≥52Number of trastuzumab-based therapiesNumber of patients015162833

[0285] In Part 1 of the study, as a result of the safety analysis at the time of data cutoff on May 24, 2024, treatment-emergent adverse events (TEAEs) were observed in 25 (78.1%) of the 32 patients, and most of the TEAEs were grade 1 or 2 in severity. Of these patients, 10 patients (31.3%) were reported to have treatment-related adverse events (TRAEs) related to the bispecific antibody AHYM12, and the most frequent TRAEs were fever and chills. No dose-limiting toxicity (DLT) occurred up to the planned highest dose of 30 mg / kg, and the maximum tolerated dose (MTD) was not reached. No dose-dependent increase in the incidence and severity of TEAEs was observed, and the bispecific antibody AHYM12 showed excellent tolerability and safety profile at all doses. From the above data, it could be seen that the bispecific antibody AHYM12 according to the present invention has very high safety without serious side effects in patients with various cancer types including biliary tract cancer.

[0286] As a result of the efficacy evaluation at the time of data cutoff on May 24, 2024, among the 31 patients who were evaluable for response, there were no complete response (CR) patients, 7 (22.6%) partial response (PR) patients, 10 (32.3%) stable disease (SD) patients, 11 (35.5%) progressive disease (PD) patients, and 3 (9.7%) not evaluable (NE) patients. As a result, it was shown that the objective response rate (ORR) was 22.6% (7 / 31 patients), and the disease control rate (DCR) was 54.8% (17 / 31 patients). The tumor types of the seven patients who reached partial response (PR) were 4 biliary tract cancer patients, 1 gastric cancer patient, 1 vaginal cancer patient, and 1 non-small cell lung cancer patient. Of these seven patients, three patients whose responses lasted more than 10 months were all patients with biliary tract cancer. The time to first response was similar at about 12 weeks regardless of dose.

[0287] As a result of comparing the antitumor activity of the bispecific antibody AHYM12 in biliary tract cancer and other cancers, partial response (PR) or stable disease (SD) was observed in 8 out of 13 patients with biliary tract cancer in whom response was evaluable, indicating that the bispecific antibody had an effective antitumor effect. In particular, 4 patients showed partial response (PR). That is, in the patients with biliary tract cancer (n=13), the ORR was confirmed to be high at 30.8% and the DCR was confirmed to be high at 61.5%. In the patients with cancers other than biliary tract cancer (n=18), the ORR was confirmed to be 16.7% and the DCR was confirmed to be 50% (see Table 12).

[0288] Summary of overall response in biliary tract cancer and other cancers in Part 1Biliary tract cancerCancers other than biliary tract cancerAll cancersComplete response (CR) (n)000Partial response (PR) (n)437Stable disease (SD) (n)4610Disease progression (PD) (n)3811Not evaluable (NE) (n)213Sum (n)131831Objective response rate (complete response + partial response)30.8% (4 / 13)16.7% (3 / 18)22.6% (7 / 31)Disease control rate (complete response + partial response + stable disease)61.5% (8 / 13)50.0% (9 / 18)54.8% (17 / 31)

[0289] Among the 13 patients with biliary tract cancer in whom response was evaluable, 0.75 mg / kg (1 patient), 1.5 mg / kg (3 patients), 3 mg / kg (1 patient), 6 mg / kg (4 patients), 12 mg / kg (2 patients), and 30 mg / kg (2 patients) were enrolled by dose level. As a result of evaluating antitumor activity in the patients with biliary tract cancer, partial response (PR) was found in 4 patients (30.8%), stable disease (SD) was found in 4 patients (30.8%), disease progression (PD) was found in 3 patients (23.1%), and not evaluable (NE) was found in 2 patients (15.4%). In patients with biliary tract cancer, the bispecific antibody AHYM12 exhibited effective therapeutic effects at doses of 1.5 mg / kg to 30 mg / kg and particularly resulted in partial response (PR) at doses of 1.5 mg / kg to 12 mg / kg (see Table 13).

[0290] Summary of dose-dependent responses in biliary tract cancer patients in Part 10.3 mpk0.75 mpk1.5 mpk3mpk6mpk12mpk20mpk30mpkTotalEvaluable patients, n0131420213Confirmed best overall responseComplete response (CR), n (%)000000000Partial response (PR), n (%)001 (33.3)1 (100)1 (25.0)1 (50.0)004 (30.8)Stable disease (SD), n (%)001 (33.3)02 (50.0)001 (50.0)4 (30.8)Disease progression (PD), n (%)01 (100)1 (33.3)00001 (50.0)3 (23.1)Not evaluable (NE), n (%)00001 (25.0)1 (50.0)002 (15.4)Objective response rate (complete response + partial response), n (%)001 (33.3)1 (100)1 (25.0)1 (50.0)004 (30.8)Disease control rate (complete response + partial response + stable disease), n (%)002 (66.7)1 (100)3 (75.0)1 (50.0)01 (50.0)8 (61.5)Time to first response (weeks), median (range)--11.6(11.6, 11.6)17.4(17.4, 17.4)11.7(11.7, 11.7)19.0(19.0, 19.0)--14.6(11.6, 19.0)

[0291] In addition, among the 13 patients with biliary tract cancer in whom response was evaluable, there were 3 HER2 IHC2+ / ISH+ biliary tract cancer patients and 10 HER2 IHC3+ biliary tract cancer patients. Among the 3 HER2 IHC2+ / ISH+ biliary tract cancer patients, 1 patient showed partial response (PR) and 2 patients showed stable disease (SD). Among the 10 HER2 IHC3+ biliary tract cancer patients, 3 patients (30%) showed partial response (PR) and 2 patients (20%) showed stable disease (SD) (see Table 14).

[0292] Summary of responses classified according to IHC in biliary tract cancer patients in Part 1Biliary tract cancer patients, nIHC2+ / ISH+Complete response (CR)0Partial response (PR)1Stable disease (SD)2Disease progression (PD)0Not evaluable (NE)0Objective response rate (complete response + partial response), n (%)1 (33.3)Disease control rate (complete response + partial response + stable disease), n (%)3 (100)IHC3+Complete response (CR)0Partial response (PR)3Stable disease (SD)2Disease progression (PD)3Not evaluable (NE)2Objective response rate (complete response + partial response), n (%)3 (30)Disease control rate (complete response + partial response + stable disease), n (%)5 (50)

[0293] From the above-described data, it was confirmed that, when the bispecific antibody AHYM12 was administered according to the method of the present invention, it exhibited a clinically significant antitumor effect in the patients with cancer, particularly biliary tract cancer, preferably HER2 IHC3+ type biliary tract cancer and / or HER2 IHC2+ / ISH+ type biliary tract cancer.

[0294] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

[0295] The present invention relates to a pharmaceutical composition containing an anti-HER2 / anti-4-1BB bispecific antibody, which may be used for the treatment of biliary tract cancer, and a method for treating biliary tract cancer using the same.

Claims

1.A pharmaceutical composition for treating biliary tract cancer in a subject, comprising, as an active ingredient, an anti-HER2 / anti-4-1BB bispecific antibody comprising a heavy chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 27, an anti-4-1BB scFv comprising the amino acid sequence of SEQ ID NO: 18, and a light chain of an anti-HER2 antibody comprising the amino acid sequence of SEQ ID NO: 28.2.The pharmaceutical composition of claim 1, wherein the subject is a subject having biliary tract cancer that is refractory or intolerant to therapy with gemcitabine, cisplatin, paclitaxel, 5-fluorouracil, leucovorin, oxaliplatin, capecitabine, irinotecan, abraxane, carboplatin, pemetrexed or a combination of two or more thereof.3.The pharmaceutical composition of claim 1, wherein the subject is a subject having biliary tract cancer that is refractory or intolerant to trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, trastuzumab rezetecan, zanidatamab (ZW25), zanidatamab zovodotin (ZW49), pertuzumab, tucatinib, lapatinib, SAR443216, BDC-1001, pembrolizumab, bevacizumab, cetuximab, lenvatinib, ramucirumab, or a combination of two or more thereof.4.The pharmaceutical composition of claim 1, wherein the subject is a subject with locally advanced or metastatic biliary tract cancer.5.The pharmaceutical composition of claim 1, wherein the biliary tract cancer has a HER2 status of IHC3+ or IHC2+ / ISH+.6.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered to the subject in an effective amount of 10 mg to 3,000 mg.7.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered to the subject in an effective amount of 0.3 mg / kg to 30 mg / kg.8.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered to the subject in an effective amount of 1.5 mg / kg to 12 mg / kg.9.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered to the subject in an effective amount of 0.3 mg / kg, 0.75 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 12 mg / kg, 20 mg / kg, or 30 mg / kg.10.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered at a frequency of once every 1, 2, 3 or 4 weeks.11.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered at a frequency of once every 3 weeks.12.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered to the subject once every three weeks at an effective amount of 0.3 mg / kg, 0.75 mg / kg, 1.5 mg / kg, 3 mg / kg, 6 mg / kg, 12 mg / kg, 20 mg / kg, or 30 mg / kg.13.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered intravenously to the subject.14.The pharmaceutical composition of claim 13, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered intravenously to the subject over 30 to 90 minutes.15.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered for at least 12 weeks.16.The pharmaceutical composition of claim 1, wherein the anti-HER2 / anti-4-1BB bispecific antibody is administered at least 4 times.17.The pharmaceutical composition of claim 1, wherein administration of the anti-HER2 / anti-4-1BB bispecific antibody to the subject delays or inhibits growth of biliary tract cancer tumors, reduces tumor cell load or tumor burden, promotes tumor regression, or causes tumor shrinkage, necrosis, or disappearance.18.The pharmaceutical composition of claim 1, wherein administration of the anti-HER2 / anti-4-1BB bispecific antibody to the subject reduces the size of the biliary tract cancer by at least 10%, 20%, 30%, 40% or 50% compared to the size before the administration.19.The pharmaceutical composition of claim 1, wherein the subject exhibits complete response (CR), partial response (PR), or stable disease (SD) after administration of the anti-HER2 / anti-4-1BB bispecific antibody.20.The pharmaceutical composition of claim 1, wherein the subject exhibits progression-free survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 18 months, at least 2 years, at least 3 years, at least 4 years or at least 5 years after initial administration of the anti-HER2 / anti-4-1BB bispecific antibody.