Pharmaceutical composition for treating or preventing cancer with low HER2 expression level

Abstract

Pharmaceutical compositions and methods are provided for the treatment and / or prevention of HER2 under-expressing and / or FcγRI-expressing cancers using an anti-4-1BB / anti-HER2 bispecific antibody.

Description

[Technical field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0002] This application claims priority to Korean Patent Application No. 10-2022-0043555, filed on April 7, 2022, the entire disclosure of which is incorporated herein by reference.

[0003] Pharmaceutical compositions and methods are provided for the treatment and / or prevention of HER2 under-expressing and / or FcγRI-expressing cancers using an anti-4-1BB / anti-HER2 bispecific antibody. [Background technology]

[0004] 4-1BB protein is a member of the TNF receptor superfamily (TNFRSF) and a costimulatory molecule expressed after immune cell activation of both innate and adaptive immune cells. 4-1BB plays an important role in regulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation and survival, cytokine secretion, and cytolytic activity of CD8 T cells. Many other studies have shown that activation of 4-1BB enhances immune responses to eliminate tumors in mice, suggesting that 4-1BB is a promising target molecule in cancer immunology. Despite its antitumor activity, anti-4-1BB antibodies caused severe hepatotoxicity in clinical applications.

[0005] On the other hand, the HER2 protein is a member of the epidermal growth factor receptor (EGFR) family and is involved in various tumor-related mechanisms. HER2 is a classical receptor tyrosine kinase (RTK) on the cell surface that induces cancer cell proliferation, invasion, and angiogenesis. Most of the HER2-targeted drugs developed so far have shown anticancer effects against cancers with high HER2 expression levels, but do not show significant therapeutic effects against cancers with low HER2 expression levels.

[0006] Therefore, for more efficient cancer treatment, it is necessary to develop drugs that are independent of the HER2 expression level, have anticancer effects against a wide range of cancers, and have low hepatotoxicity. Summary of the Invention [Problem to be solved by the invention]

[0007] In one embodiment, there is provided a pharmaceutical composition for the prevention and / or treatment of cancer comprising an anti-HER2 / anti-4-1BB bispecific antibody, (1) an anti-HER2 antibody or antigen-binding fragment thereof as a HER2 targeting moiety capable of specifically recognizing and / or binding to HER2 protein; (2) An anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety capable of specifically recognizing and / or binding to 4-1BB protein. A pharmaceutical composition comprising:

[0008] The cancer can be a cancer characterized by low expression of HER2 (HER2 low expression), expression of FcγRI, or both.

[0009] In another embodiment, a method for preventing or treating cancer is provided, comprising administering a pharma- tically effective amount of said bispecific antibody or said pharmaceutical composition to a subject in need of said cancer prevention or treatment. The method may further comprise the step of identifying a subject in need of said cancer prevention or treatment prior to said administering step. The cancer may be a cancer characterized by low expression of HER2, FcγRI expression, or both.

[0010] In one embodiment, the bispecific antibody or the pharmaceutical composition is provided for use in the prevention or treatment of cancer. In another embodiment, the use of the bispecific antibody in the preparation of a pharmaceutical composition for the prevention or treatment of cancer is provided. The cancer may be a cancer characterized by low expression of HER2, FcγRI expression, or both. [Means for solving the problem]

[0011] The present disclosure relates to a bispecific antibody comprising an antibody specific to a tumor-associated antigen (TAA; HER2) and an antibody specific to 4-1BB, and uses thereof. The bispecific antibody can activate 4-1BB signaling and enhance immune responses against HER2-high expressing tumors (cancers) and cancer cells with low HER2 expression and / or FcγRI expression characteristics. Thus, the bispecific antibody provided herein can be used as a cancer immunotherapy agent exhibiting anti-cancer activity against HER2-high expressing tumors (cancers) and cancers characterized by low HER2 expression and / or FcγRI expression.

[0012] Provided herein is an anti-cancer (including preventative, therapeutic, ameliorative, alleviative, and / or curative) use of an anti-HER2 / anti-4-1BB bispecific antibody in cancers with low HER2 expression and / or FcγRI expression characteristics, the anti-HER2 / anti-4-1BB bispecific antibody comprising: (1) an anti-HER2 antibody or antigen-binding fragment thereof as a HER2 targeting moiety capable of specifically recognizing and / or binding to HER2 protein; (2) An anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety capable of specifically recognizing and / or binding to a 4-1BB protein. may include. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will now be described in more detail.

[0014] [Terminology definition] As used herein, "consisting of a certain sequence," "consisting essentially of a certain sequence," or "comprising a certain sequence" may mean any case in which the certain sequence is included, but is not intended to exclude cases in which the certain sequence includes additional sequences other than the certain sequence.

[0015] As used herein, the terms "a protein or polypeptide comprising or consisting of an amino acid sequence identified by a SEQ ID NO" and "a gene or polynucleotide comprising or consisting of a nucleic acid sequence identified by a SEQ ID NO" may mean a protein (or polypeptide) or a gene (or polynucleotide) that consists essentially of an amino acid sequence or a nucleic acid sequence or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity to said amino acid sequence or nucleic acid sequence while retaining its inherent and / or intended activity and / or function.

[0016] As used herein, the term "antibody" can encompass a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with their several subclasses (e.g., γ1-γ4), and light chains are classified as kappa or lambda (K, λ). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to provide functional specialization.

[0017] An intact antibody contains two full-length light chains and two full-length heavy chains, and each light chain may be linked to a heavy chain by a disulfide bond. An antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region may be of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further classified as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constant region may be of kappa (κ) or lambda (λ) type.

[0018] The term "heavy chain" refers to a variable region V that contains sufficient amino acid sequence to provide specificity for an antigen. H and three constant regions C H1 , C H2 and C H3 The term "light chain" may refer to a full-length heavy chain or a fragment thereof, including the variable region V that contains sufficient amino acid sequence to provide specificity for an antigen. L and constant region C L It may refer to a full length light chain or a fragment thereof, comprising:

[0019] The term "complementarity determining region (CDR)" refers to an amino acid sequence found in the hypervariable region of an immunoglobulin heavy or light chain. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDRs may provide residues that play a role in the binding of an antibody to an antigen or epitope. The terms "specifically bind" or "specifically recognized" are well known in the art and refer to the specific interaction of an antibody and an antigen to induce immunological activity.

[0020] In this disclosure, antibodies can include, but are not limited to, polyclonal or monoclonal; and / or human, humanized, animal (e.g., mouse, rabbit, etc.) derived antibodies, or chimeric antibodies (e.g., mouse-human chimeric antibodies).

[0021] Animal-derived antibodies, which are produced by immunizing animals with a desired antigen, may cause immune rejection when administered to humans, usually for therapeutic purposes, and therefore chimeric antibodies have been developed to suppress such immune rejection. Chimeric antibodies are formed by replacing the constant region of an animal-derived antibody, which causes an anti-isotype reaction, with the constant region of a human antibody using genetic engineering techniques. Although chimeric antibodies have significantly improved anti-isotype reactions compared to animal-derived antibodies, potential side effects due to anti-idiotype reactions still exist due to the presence of animal-derived amino acids in the variable region. Humanized antibodies have been developed to improve these side effects. They are produced by transplanting the CDRs (complementarity determining regions), which play an important role in antigen binding, from the variable region of a chimeric antibody into a human antibody framework.

[0022] As used herein, the term "antigen-binding fragment" refers to a fragment derived from the complete immunoglobulin structure that contains a portion capable of binding to an antigen, such as a CDR. For example, the antigen-binding fragment may be, but is not limited to, an scFv, (scFv)2, scFv-Fc, Fab, Fab', or F(ab')2. For example, in the present disclosure, the antigen-binding fragment may be at least one antibody-derived fragment containing a CDR selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2.

[0023] Among the antigen-binding fragments, Fab, which has a structure consisting of light chain and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), has one antigen-binding site.

[0024] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are formed by disulfide bond formation between the cysteine ​​residues in the hinge region of Fab'.

[0025] Fv is the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are well known in the art. A two-chain Fv may have a structure in which a heavy chain variable region and a light chain variable region are non-covalently linked, and a single-chain Fv (scFv) may generally have a dimeric structure like a two-chain Fv in which a heavy chain variable region and a light chain variable region are covalently linked via a peptide linker or directly linked to each other at their C-terminus.

[0026] Antigen-binding fragments can be obtained using proteases (e.g., digestion of whole antibodies with papain to produce Fab fragments or with pepsin to produce F(ab')2 fragments) or can be prepared by recombinant techniques.

[0027] The immunoglobulin (e.g., human immunoglobulin) or antibody molecule herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.), class (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, IgA1, IgA2, etc.), or subclass of immunoglobulin molecule.

[0028] In the antibody or antibody fragment, portions other than the CDRs or variable regions (e.g., the constant region) may be derived from a human antibody, in particular, they may be derived from IgG, such as IgG1, IgG2, IgG3, or IgG4, IgA, IgD, IgE, IgM, or IgY.

[0029] The antibody or antigen-binding fragment can be chemically or recombinantly synthesized (non-naturally occurring).

[0030] [4-1BB target part] The anti-HER2 / anti-4-1BB bispecific antibody may comprise an anti-4-1BB antibody or an antigen-binding fragment thereof as the 4-1BB targeting moiety.

[0031] The term "4-1BB", also known as CD137 or TNF receptor superfamily member 9 (TNFRSF9), is a member of the TNF receptor superfamily (TNFRSF) and is a costimulatory molecule expressed after immune cell activation of both innate and adaptive immune cells. 4-1BB plays an important role in regulating the activity of various immune cells. As used herein, 4-1BB may be derived from a mammal, such as Homo sapiens (human) (NCBI Accession No. NP_001552.2). For example, the human 4-1BB protein (NP_001552.2) may be represented by the following amino acid sequence (SEQ ID NO:89): [ka]

[0032] In one example, the anti-4-1BB antibody or antigen-binding fragment thereof is Complementarity determining region (CDR)-H1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3; H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 5, or 6; H-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, or 11; L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or 13; L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or 15; and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or 17 may include.

[0033] The amino acid sequences of the CDRs of the anti-4-1BB antibodies or antigen-binding fragments are illustrated in Table 1: [Table 1]

[0034] For example, the anti-4-1BB antibody or antigen-binding fragment thereof may be H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; H-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; or H-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, H-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, L-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. may include.

[0035] In another example, the anti-4-1BB antibody or antigen-binding fragment thereof is A heavy chain variable region comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 5 or 6, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, 8, 9, 10 or 11; and a light chain variable region comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or 13, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or 15, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or 17. may include.

[0036] In another example, the anti-4-1BB antibody or antigen-binding fragment thereof is a heavy chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29; and a light chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, or 88. may include.

[0037] The amino acid sequences of the variable regions of the anti-4-1BB antibodies or antigen-binding fragments are illustrated in Table 2: [Table 2] TIFF2025512915000005.tif88170

[0038] For example, the anti-4-1BB antibody or antigen-binding fragment thereof may be a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; or A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88. may include.

[0039] The framework amino acid sequences of the variable regions of anti-4-1BB antibodies or antigen-binding fragments are illustrated in Table 3: [Table 3]

[0040] In another example, an anti-4-1BB antibody or antigen-binding fragment thereof may comprise a heavy chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO:56, 57, 58, 59, 60, or 61; and a light chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO:62, 63, or 64.

[0041] For example, the anti-4-1BB antibody or antigen-binding fragment thereof may be a heavy chain comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:56, 57, 58, 59, 60, or 61; and a light chain comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:62; a heavy chain comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:56, 57, 58, 59, 60, or 61; and a light chain comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:63; or a heavy chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, 60, or 61; and a light chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 64. may include.

[0042] In another example, the anti-4-1BB antibody or antigen-binding fragment thereof is a heavy chain variable region comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 5, or 6, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, or 11; A light chain variable region comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or 13, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or 15, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or 17. and the scFv (single chain variable fragment) comprising Here, the heavy chain variable region and the light chain variable region can be linked to each other in any order, either directly (eg, without a linker) or via a peptide linker.

[0043] In one example, the anti-4-1BB scFv is a heavy chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29; A light chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, or 88. It may include, Here, the heavy chain variable region and the light chain variable region may be linked to each other directly or via a peptide linker in any order.

[0044] In one example, the anti-4-1BB scFv is a heavy chain variable region comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:24, 25, 26, 27, 28, or 29, and a light chain variable region comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:33; a heavy chain variable region comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:24, 25, 26, 27, 28, or 29, and a light chain variable region comprising, or consisting essentially of, the amino acid sequence of SEQ ID NO:34; or a heavy chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, or 29, and a light chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 88. It may include, Here, the heavy chain variable region and the light chain variable region may be linked to each other directly or via a peptide linker in any order.

[0045] In the present disclosure, an anti-4-1BB scFv comprises a heavy chain variable region and a light chain variable region in any order. For example, an anti-4-1BB scFv may comprise a light chain variable region and a heavy chain variable region in the N-terminal to C-terminal direction. Alternatively, an anti-4-1BB scFv may comprise a heavy chain variable region and a light chain variable region in the N-terminal to C-terminal direction. In one example, an anti-4-1BB scFv may comprise a light chain variable region, a peptide linker, and a heavy chain variable region in the N-terminal to C-terminal direction. In another example, an anti-4-1BB scFv may comprise a heavy chain variable region, a peptide linker, and a light chain variable region in the N-terminal to C-terminal direction.

[0046] [HER2 target part] The anti-HER2 / anti-4-1BB bispecific antibody may comprise an anti-HER2 antibody or an antigen-binding fragment thereof as the HER2 targeting moiety.

[0047] "HER2 (human epidermal growth factor receptor 2)" is encoded by the ERBB2 gene and is a member of the epidermal growth factor receptor (EGFR / ErbB) family. HER2 is known to play an essential role in regulating cell proliferation and differentiation. In particular, upon binding to extracellular growth factors, it has a strong tendency to assemble into homodimers and / or heterodimers with other HER receptors, activating several forms of signal transduction pathways to induce cell death, survival, or cell proliferation. For example, the HER2 protein can be a polypeptide deposited under GenBank Accession Nos. NP_004439.2, NP_001005862.1, etc., which is encoded by a nucleotide sequence (mRNA) deposited under GenBank Accession Nos. NM_004448.4, NM_001005862.3, etc., respectively.

[0048] In one embodiment, the anti-HER2 antibody may be selected from the group consisting of trastuzumab, pertuzumab, and trastuzumab emtansine (T-DM1).

[0049] The antigen-binding region of an anti-HER2 antibody that recognizes HER2 as an antigen may be scFv, (scFv)2, Fab, Fab', or F(ab')2 of an anti-HER2 antibody selected from the group consisting of trastuzumab, pertuzumab, and trastuzumab emtansine.

[0050] The anti-HER2 antibody or antigen-binding fragment thereof can be an anti-HER2 antibody or antigen-binding fragment thereof that contains the six CDRs of trastuzumab, pertuzumab, or trastuzumab emtansine.

[0051] In another embodiment, the anti-HER2 antibody or antigen-binding fragment thereof can be trastuzumab or an antigen-binding fragment thereof, or a variant thereof.

[0052] For example, the anti-HER2 antibody or antigen-binding fragment thereof is H-CDR1 comprising the amino acid sequence of SEQ ID NO: 65; H-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; H-CDR3 comprising the amino acid sequence of SEQ ID NO: 67; L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68; L-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70 may include.

[0053] The amino acid sequences of the CDRs of the anti-HER2 antibodies or antigen-binding fragments are illustrated in Table 4: [Table 4]

[0054] In another example, the anti-HER2 antibody, or antigen-binding fragment thereof, A heavy chain variable region comprising H-CDR1 comprising the amino acid sequence of SEQ ID NO: 65, H-CDR2 comprising the amino acid sequence of SEQ ID NO: 66, and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 67; and a light chain variable region comprising L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70. may include.

[0055] In another example, the anti-HER2 antibody, or antigen-binding fragment thereof, a heavy chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO:71; and a light chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO:72. may include.

[0056] The amino acid sequences of the variable regions of the anti-HER2 antibodies or antigen-binding fragments are illustrated in Table 5: [Table 5]

[0057] In another embodiment, the anti-HER2 antibody, or antigen-binding fragment thereof, a heavy chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 73 or 74; and a light chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 75. may include.

[0058] In another embodiment, the anti-HER2 antibody, or antigen-binding fragment thereof, a heavy chain variable region comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 65, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 66, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 67; A light chain variable region comprising L-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, L-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 70. and the scFv (single chain variable fragment) comprising Here, the heavy chain variable region and the light chain variable region can be linked to each other in any order, either directly (eg, without a linker) or via a peptide linker.

[0059] In another embodiment, the anti-HER2 antibody, or antigen-binding fragment thereof, a heavy chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO:71; and a light chain variable region comprising or consisting essentially of the amino acid sequence of SEQ ID NO:72. may be a scFv (single chain variable fragment) comprising: Here, the heavy chain variable region and the light chain variable region may be linked to each other directly or via a peptide linker in any order.

[0060] In the present disclosure, the anti-HER2 scFv may comprise a heavy chain variable region and a light chain variable region in any order. For example, the anti-HER2 scFv may comprise a light chain variable region and a heavy chain variable region in the N-terminal to C-terminal direction. Alternatively, the anti-HER2 scFv may comprise a heavy chain variable region and a light chain variable region in the N-terminal to C-terminal direction. In one example, the anti-HER2 scFv may comprise a light chain variable region, a peptide linker, and a heavy chain variable region in the N-terminal to C-terminal direction. In another example, the anti-HER2 scFv may comprise a heavy chain variable region, a peptide linker, and a light chain variable region in the N-terminal to C-terminal direction.

[0061] [Bispecific antibody] The present disclosure relates to (1) an anti-HER2 antibody or antigen-binding fragment thereof as a HER2 targeting moiety capable of specifically recognizing and / or binding to a HER2 protein; (2) An anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety capable of specifically recognizing and / or binding to a 4-1BB protein. The present invention provides an anti-HER2 / anti-4-1BB bispecific antibody comprising:

[0062] The anti-HER2 / anti-4-1BB bispecific antibody may activate 4-1BB signaling only when cross-linked by HER2-expressing tumor cells. Furthermore, the anti-4-1BB antibody or antigen-binding fragment thereof comprised in the bispecific antibody may be characterized by localization and / or activation only in the tumor microenvironment (TME) and / or significantly reduced hepatotoxicity compared to conventional anti-4-1BB antibodies, while maintaining an enhanced immune response and / or efficacy in tumor therapy.

[0063] In one embodiment, a bispecific antibody may comprise a full-length anti-HER2 antibody and an antigen-binding fragment (e.g., scFv) of an anti-4-1BB antibody, where the antigen-binding fragment of the anti-4-1BB may be linked directly or via a peptide linker to the N-terminus, C-terminus, or both of the full-length anti-HER2 antibody. In another embodiment, a bispecific antibody may comprise a full-length anti-4-1BB antibody and an antigen-binding fragment (e.g., scFv) of an anti-HER2 antibody, where the antigen-binding fragment of the anti-HER2 antibody may be linked directly or via a peptide linker to the N-terminus, C-terminus, or both of the full-length anti-4-1BB antibody.

[0064] In one embodiment, the scFv in the bispecific antibody may comprise a heavy chain variable region and a light chain variable region in any order, for example, the scFv in the bispecific antibody may comprise a light chain variable region and a heavy chain variable region in the N-terminal to C-terminal direction, optionally including a peptide linker between them, or the scFv in the bispecific antibody may comprise a heavy chain variable region and a light chain variable region in the N-terminal to C-terminal direction, optionally including a peptide linker between them.

[0065] When the bispecific antibody comprises a full-length anti-HER2 antibody and an anti-4-1BB scFv, the bispecific antibody comprises: (i) from N-terminus to C-terminus, a heavy chain of an anti-HER2 antibody; optionally a peptide linker (first peptide linker), and a first polypeptide comprising an anti-4-1BB scFv; (ii) a second polypeptide comprising a light chain of an anti-HER2 antibody. It may include, Here, the anti-4-1BB scFv is composed of the following in the N-terminal to C-terminal direction: The light chain variable region of an anti-4-1BB antibody, Optionally, a peptide linker (second peptide linker), and Heavy chain variable region of anti-4-1BB antibody may include.

[0066] Alternatively, the bispecific antibody may comprise: (i) from the N-terminus to the C-terminus, anti-4-1BB scFv; Optionally, a peptide linker (first peptide linker), and a first polypeptide comprising a heavy chain of an anti-HER2 antibody; (ii) a second polypeptide comprising a light chain of an anti-HER2 antibody. It may include, Here, the anti-4-1BB scFv is composed of the following in the N-terminal to C-terminal direction: The light chain variable region of an anti-4-1BB antibody, Optionally, a peptide linker (second peptide linker), and Heavy chain variable region of anti-4-1BB antibody may include.

[0067] Alternatively, the bispecific antibody may comprise: (i) from N-terminus to C-terminus, a heavy chain of an anti-HER2 antibody; optionally a peptide linker (first peptide linker), and a first polypeptide comprising an anti-4-1BB scFv; (ii) a second polypeptide comprising a light chain of an anti-HER2 antibody. It may include, Here, the anti-4-1BB scFv is composed of the following in the N-terminal to C-terminal direction: A heavy chain variable region of an anti-4-1BB antibody, Optionally, a peptide linker (second peptide linker), and Anti-4-1BB antibody light chain variable region may include.

[0068] Alternatively, the bispecific antibody may comprise: (i) in the N-terminal to C-terminal direction, anti-4-1BBscFv, optionally a peptide linker (first peptide linker), and Anti-HER2 antibody heavy chain and a first polypeptide comprising: (ii) a second polypeptide comprising a light chain of an anti-HER2 antibody. It may include, Here, the anti-4-1BB scFv is composed of the following in the N-terminal to C-terminal direction: A heavy chain variable region of an anti-4-1BB antibody, Optionally, a peptide linker (second peptide linker), and Anti-4-1BB antibody light chain variable region may include.

[0069] When the bispecific antibody comprises a full-length anti-4-1BB antibody and an anti-HER2 scFv, the bispecific antibody comprises: (i) in the N-terminal to C-terminal direction, The heavy chain of an anti-4-1BB antibody, optionally a peptide linker (first peptide linker), and a first polypeptide comprising an anti-HER2 scFv; (ii) a second polypeptide comprising the light chain of an anti-4-1BB antibody; It may include, Here, the anti-HER2 scFv is composed of the following in the N-terminal to C-terminal direction: The light chain variable region of an anti-HER2 antibody, Optionally, a peptide linker (second peptide linker), and Heavy chain variable region of anti-HER2 antibody may include.

[0070] Alternatively, the bispecific antibody may comprise: (i) in the N-terminal to C-terminal direction, anti-HER2 scFv; Optionally, a peptide linker (first peptide linker), and Anti-4-1BB antibody heavy chain and a first polypeptide comprising: (ii) a second polypeptide comprising the light chain of an anti-4-1BB antibody; It may include, Here, the anti-HER2 scFv is, in the N-terminal to C-terminal direction, The light chain variable region of an anti-HER2 antibody, Optionally, a peptide linker (second peptide linker), and Heavy chain variable region of anti-HER2 antibody may include.

[0071] Alternatively, the bispecific antibody may comprise: (i) in the N-terminal to C-terminal direction, The heavy chain of an anti-4-1BB antibody, Optionally, a peptide linker (first peptide linker), and Anti-HER2 scFv and a first polypeptide comprising: (ii) a second polypeptide comprising the light chain of an anti-4-1BB antibody; It may include, Here, the anti-HER2 scFv is, in the N-terminal to C-terminal direction, A heavy chain variable region of an anti-HER2 antibody, Optionally, a peptide linker (second peptide linker), and Anti-HER2 antibody light chain variable region may include.

[0072] Alternatively, the bispecific antibody may comprise: (i) in the N-terminal to C-terminal direction, anti-HER2 scFv, Optionally, a peptide linker (first peptide linker), and Anti-4-1BB antibody heavy chain and a first polypeptide comprising: (ii) a second polypeptide comprising the light chain of an anti-4-1BB antibody; It may include, Here, the anti-HER2 scFv is composed of the following in the N-terminal to C-terminal direction: A heavy chain variable region of an anti-HER2 antibody, Optionally, a peptide linker (second peptide linker), and Anti-HER2 antibody light chain variable region may include.

[0073] The first peptide linker and the second peptide linker may or may not be independently present in the bispecific antibody and may be the same or different from each other.

[0074] In one embodiment, the anti-HER2 / anti-4-1BB bispecific antibody comprises (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, or 84; (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 75 may include.

[0075] For example, the anti-HER2 / anti-4-1BB bispecific antibody (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 83; (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 75 may include.

[0076] In another embodiment, both the HER2 targeting moiety and the 4-1BB targeting moiety in the bispecific antibody can be full length antibodies or antigen-binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof, and can be linked to each other via a peptide linker or directly.

[0077] Because each antibody can bind both 4-1BB (e.g., human 4-1BB) and HER2 (e.g., human HER2), the CDR sequences, or VH (variable heavy chain) and VL (variable light chain) sequences disclosed herein can be "mixed and matched" to create different anti-HER2 / anti-4-1BB bispecific molecules.

[0078] [Peptide linker] In highly purified antibodies, the bispecific antibody may comprise a peptide linker between the heavy chain of the first polypeptide and the scFv (first peptide linker) and / or between the variable regions of the heavy and light chains of the scFv (second peptide linker).

[0079] As used herein, the term "peptide linker" may refer to an oligopeptide containing 1 to 100 amino acids, particularly 2 to 50 amino acids, where each amino acid may be any type of amino acid without limitation. Any common peptide linker, with or without appropriate modification, may be used to achieve a particular purpose. In certain embodiments, the peptide linker may contain, for example, Gly, Asn, and / or Ser residues, and / or may contain neutral amino acids such as Thr and / or Ala. Amino acid sequences suitable for peptide linkers may be known in the relevant technical field. The length of the peptide linker may be appropriately determined to the extent that it does not affect the function of the polypeptide and / or scFv. For example, the peptide linker can be from about 1 to about 100 amino acids, from about 2 to about 50 amino acids, or from about 5 to about 25 amino acids (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), each of which can be independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. m S l ) n (where m, l, and n are "G", "S", and "(G m S l )" each of which may be independently selected from an integer from about 1 to about 10, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker may be, but is not limited to, a peptide of (GGGGS)2 (SEQ ID NO:90), (GGGGS)3 (SEQ ID NO:85), (GGGGS)4 (SEQ ID NO:87), or (GS)9 (SEQ ID NO:87).

[0080] [Medicinal uses] The pharmaceutical use of the bispecific antibody for the prevention and / or treatment of cancer is provided. More specifically, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody as an active ingredient. The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier. The pharmaceutical composition may further comprise a cancer immunotherapeutic agent. The bispecific antibody and the cancer immunotherapeutic agent may be included together in the form of a fusion protein or a multispecific antibody (such as a triabody or a tetrabody), or may be formulated for co-administration. In one embodiment, the bispecific antibody and the cancer immunotherapeutic agent may be formulated in a single formulation, or the bispecific antibody and the cancer immunotherapeutic agent may be formulated separately and then mixed, but are not limited thereto. The pharmaceutical composition may be used for the prevention and / or treatment of cancer.

[0081] In another embodiment, a pharmaceutical composition for the prevention and / or treatment of cancer is provided, comprising a bispecific antibody as an active ingredient. The pharmaceutical composition may further comprise a cancer immunotherapeutic agent. The bispecific antibody and the cancer immunotherapeutic agent may be included together in the form of a fusion protein or a multispecific antibody (such as a triabody or a tetrabody), or may be formulated for simultaneous administration. In one embodiment, the bispecific antibody and the cancer immunotherapeutic agent may be formulated in a single formulation, or the bispecific antibody and the cancer immunotherapeutic agent may be formulated separately and then mixed, but are not limited thereto.

[0082] In another embodiment, a method for preventing and / or treating cancer is provided, comprising administering a pharma- ceutically effective amount of a bispecific antibody or pharmaceutical composition to a subject in need thereof. The method may further comprise identifying a subject in need of cancer prevention and / or treatment prior to the administering step. The method may further comprise administering a pharma- ceutically effective amount of a cancer immunotherapeutic agent to the subject. The steps of administering the bispecific antibody and administering the cancer immunotherapeutic agent may be performed sequentially in any order or simultaneously. The bispecific antibody and the cancer immunotherapeutic agent may be formulated and administered as a single formulation or may be formulated separately and then administered simultaneously or sequentially in any order, including but not limited to the above. Furthermore, the cancer immunotherapeutic agent may be administered in the form of a fusion protein or a multispecific antibody (such as, for example, a triabody or a tetrabody) together with the bispecific antibody, including but not limited to the above.

[0083] In another embodiment, there is provided a use of a bispecific antibody or a pharmaceutical composition in the prevention and / or treatment of cancer. In another embodiment, there is provided a use of a bispecific antibody in the preparation of a pharmaceutical composition for the prevention and / or treatment of cancer.

[0084] In another embodiment, a pharmaceutical composition for simultaneous administration for the prevention and / or treatment of cancer is provided, comprising a bispecific antibody and a cancer immunotherapeutic agent as active ingredients. The pharmaceutical composition for simultaneous administration may further comprise a pharma- ceutically acceptable carrier. The bispecific antibody and the cancer immunotherapeutic agent may be included together in the form of a fusion protein or a multispecific antibody (e.g., a triabody or a tetrabody, etc.), or may be formulated for simultaneous administration. In one embodiment, the bispecific antibody and the cancer immunotherapeutic agent may be formulated in a single formulation, or the bispecific antibody and the cancer immunotherapeutic agent may be formulated separately and then mixed, but are not limited thereto.

[0085] In another embodiment, a method for preventing and / or treating cancer is provided, comprising administering a pharma- ceutical effective amount of a bispecific antibody and a pharma- ceutical effective amount of a cancer immunotherapeutic agent to a subject in need thereof. In this method, the pharma- ceutical effective amount of the bispecific antibody and the pharma- ceutical effective amount of the cancer immunotherapeutic agent can be administered simultaneously or sequentially in any order. The bispecific antibody and the cancer immunotherapeutic agent can be formulated and administered as a single formulation, or can be formulated separately and then administered simultaneously or sequentially in any order, including but not limited to the above. Furthermore, the cancer immunotherapeutic agent can be administered in the form of a fusion protein or a multispecific antibody (such as, for example, a triabody or a tetrabody) together with the bispecific antibody, including but not limited to the above.

[0086] In another embodiment, the use of a bispecific antibody and a cancer immunotherapeutic agent in the prevention and / or treatment of cancer is provided. In another embodiment, the use of a bispecific antibody and a cancer immunotherapeutic agent in the preparation of a pharmaceutical composition for the prevention and / or treatment of cancer is provided. The bispecific antibody and the cancer immunotherapeutic agent may be included together in the form of a fusion protein or a multispecific antibody (such as a triabody or a tetrabody) or may be formulated for co-administration. In one example, the bispecific antibody and the cancer immunotherapeutic agent may be formulated in a single formulation, or the bispecific antibody and the cancer immunotherapeutic agent may be formulated separately and then mixed, but are not limited thereto.

[0087] As used herein, the cancer immunotherapeutic agent may be selected from any agent that exerts its anti-cancer effect by activating the immune system, for example by inhibiting immune checkpoints. For example, the cancer immunotherapeutic agent may be an agent (e.g., an antibody, etc.) that inhibits one or more activities (e.g., interactions with respective binding proteins (e.g., ligands, etc.)) selected from the group consisting of PD-1, PD-L1, TIGIT, 4-1BB, OX40, CTLA-4, LAG-3, TIM-3, GITR, GITRL, ICOS, ICOSL, and VISTA. In one embodiment, the cancer immunotherapeutic agent may be an agent that inhibits the interaction between PD-1 and PD-L1, such as, but not limited to, an agent that targets PD-1 (PD-1 inhibitor), an agent that targets PD-L1 (PD-L1 inhibitor), or a combination thereof. The agent targeting PD-1 may be one or more selected from the group consisting of, but is not limited to, a protein (e.g., an antibody, an antigen-binding fragment of an antibody, an antibody analog, etc.) that binds (e.g., specifically binds) to PD-1, a nucleic acid molecule (e.g., an aptamer, siRNA, shRNA, microRNA, etc.), a small molecule compound, etc. Furthermore, the agent targeting PD-L1 may be one or more selected from the group consisting of, but is not limited to, a protein (e.g., an antibody, an antigen-binding fragment of an antibody, an antibody analog, etc.) that binds (e.g., specifically binds) to PD-L1, a nucleic acid molecule (e.g., an aptamer, siRNA, shRNA, microRNA, etc.), a small molecule compound, etc.

[0088] In one embodiment, the cancer immunotherapeutic agent may be at least one selected from the group consisting of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, an antigen-binding fragment thereof, etc.) and a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody, an antigen-binding fragment thereof).

[0089] For example, the PD-1 inhibitor may be an anti-PD-1 antibody or an antigen-binding fragment thereof, and the anti-PD-1 antibody may be one or more selected from the group consisting of, but not limited to, pembrolizumab, nivolumab, cemiplimab, dostarlimab, etc. The PD-L1 inhibitor may be an anti-PD-L1 antibody or an antigen-binding fragment thereof, and the anti-PD-L1 antibody may be one or more selected from the group consisting of, but not limited to, atezolizumab, durvalumab, avelumab, etc.

[0090] The cancer can be a cancer characterized by low expression of HER2, expression of FcγRI, or both. For example, the cancer can be a HER2 low-expressing cancer.

[0091] As used herein, the term "HER2 low-expressing cancer" is not particularly limited as long as it is recognized as a HER2 low-expressing cancer by those skilled in the art, but may be broadly interpreted to include any case in which the expression of HER2 in a tumor as a whole is below a certain level. For example, it may mean that HER2 is expressed at a low level, that the percentage of cells expressing HER2 in a tumor is low, or that HER2 is not expressed (HER2 naive). In another embodiment, when interpreted narrowly, "HER2 low-expressing tumor (cancer)" may be interpreted to mean, but is not limited to, that HER2 is expressed at a low level, or that the percentage of cells expressing HER2 in a tumor is low.

[0092] In one embodiment, HER2 low expressing cancer may refer to one or more of the following: (1) IHC score of 0, 1+, or 2+ as measured by immunohistochemistry using an anti-HER2 antibody specific for HER2; (2) The HER2 level in the tumor, as measured by a common protein measurement method, is 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less (the lower limit can be 0% or more than 0%) compared to the HER2 level in an SK-BR-3 cell line (e.g., HTB-30 (ATCC)); (3) The HER2 level in the tumor, as measured by a common protein measurement method, is lower than the level in the HCC1954 cell line (e.g., CRL-2338 (ATCC)) (the lower limit can be zero or a value greater than zero); and (4) The percentage of tumor cells expressing HER2 among all tumor cells constituting the cancer is 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less based on cell number, volume, or weight (the lower limit may be 0% or a number greater than 0%).

[0093] In another embodiment, the "HER2 low-expressing cancer" may be (i) a cancer in which HER2 expression is determined to be 2+ by immunohistochemistry and HER2 expression is determined to be negative by in situ hybridization (ISH), or (ii) a cancer in which HER2 expression is determined to be 1+ by immunohistochemistry. The ISH method may include fluorescent in situ hybridization (FISH) or dual in situ hybridization (DISH). Any method of determining HER2 expression by immunohistochemistry, or any method of determining positive or negative HER2 expression by ISH, may be used without limitation as long as it is recognized by those skilled in the art. For example, any method of determining HER2 expression based on the ASCO / CAP guideline, NCCN guideline, etc. may be used without limitation. In another embodiment, the "HER2 low-expressing cancer" may be, but is not limited to, a cancer recognized by those skilled in the art as a HER2 low-expressing cancer by next-generation sequencing (NGS).

[0094] In one example, a HER2 low-expressing cancer can mean that the percentage of tumor cells that express HER2 out of all tumor cells that make up the 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 cell number, volume, or weight.

[0095] Common protein measurement methods may be measurements by common enzyme reactions, fluorescence, luminescence, and / or radiation detection using compounds, antibodies, aptamers, etc. that specifically bind to the protein to be measured (e.g., HER2), and may include, but are not limited to, immunohistochemistry, ISH, immunochromatography, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), luminescence immunoassay (LIA), western blotting, microarrays, etc.

[0096] FcγRI refers to Fc gamma receptor I, also known as CD64. FcγRI-expressing cancer can refer to any type of cancer that contains cells that express FcγRI (e.g., infiltrating immune cells in a tumor).

[0097] The anti-HER2 / anti-4-1BB bispecific antibody provided herein is characterized by having anti-cancer activity in HER2-high and HER2-low expressing cancers, having excellent 4-1BB activation and / or immune response-inducing activity in FcγRI-expressing cancers, and providing excellent anti-cancer effects in cancers characterized by low HER2 expression and / or FcγRI expression.

[0098] The cancer that may be prevented and / or treated by the bispecific antibody or pharmaceutical composition may be a cancer with low HER2 expression and / or FcγRI expression characteristics. The cancer may be selected from solid cancers and hematological cancers with low HER2 expression and / or FcγRI expression characteristics. The cancer may have HER2 low expression and / or FcγRI expression characteristics and may be one or more selected from the group consisting of, but not limited to, breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell carcinoma of the lung, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, skin or ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, liver cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatocellular adenoma, endometrial or uterine cancer, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, etc. The cancer may be a primary cancer, a metastatic cancer, or a recurrent cancer.

[0099] As used herein, the term "cancer prevention and / or treatment" can mean killing cancer cells, inhibiting the growth of cancer cells, alleviating symptoms associated with cancer, inhibiting the spread of cancer, or inhibiting the recurrence of cancer.

[0100] As used herein, the term "enhanced immune response" can mean, but is not limited to, 4-1BB signal activation, enhancing or strengthening an immune response associated with 4-1BB, such as 4-1BB-induced signal activation (e.g., 4-1BB-induced NF-kB signal activation, increased cytokine release, killing of target cells by immune cells such as T cells, etc.). In one embodiment, the enhanced immune response by the bispecific antibodies provided herein can occur in the presence of HER2.

[0101] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, diluent, and / or additive in addition to the bispecific antibody as an active ingredient. The pharma- ceutically acceptable carrier, diluent, and / or additive may be any selected from those commonly used in the formulation of antibodies. For example, pharma- ceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0102] The pharmaceutical composition may further comprise one or more selected from the group consisting of lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, preservatives, and the like.

[0103] The bispecific antibody or pharmaceutical composition may be administered orally or parenterally to a subject. Parenteral administration may be intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, topical, intranasal, intrapulmonary, or rectal. Since oral administration may lead to digestion of proteins or peptides, the active ingredient in a composition for oral administration may be coated or formulated to prevent digestion in the stomach. In addition, the composition may be administered using any device that allows the active ingredient to be delivered to target cells (e.g., cancer cells).

[0104] As used herein, the term "pharmacologically effective amount" may refer to an amount in which the active ingredient, bispecific antibody and / or cancer immunotherapeutic agent can exert a pharma- tically meaningful effect in the prevention or treatment of cancer. A pharma- tically effective amount of a bispecific antibody and / or cancer immunotherapeutic agent, or a suitable dosage of a pharmaceutical composition expressed as an amount of a bispecific antibody, may be formulated in various ways depending on various factors such as the patient's age, body weight, sex, medical condition, diet, excretion rate, and / or response sensitivity, type of formulation, administration time, administration route, and administration method. For example, a pharma- tically effective amount of a bispecific antibody (and / or cancer immunotherapeutic agent) in an adult, or a suitable dosage of a pharmaceutical composition, may be about 0.001 to about 1000 mg (amount of bispecific antibody) / kg (body weight) / day, about 0.01 to about 100 mg / kg / day, or about 0.1 to about 50 mg / kg / day.

[0105] The subject to which the bispecific antibody and / or cancer immunotherapeutic agent, or pharmaceutical composition is administered may be a mammal, such as, but not limited to, a human, monkey, rat, mouse, dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, cow, or a cell or tissue obtained therefrom, and the subject may be suffering from cancer.

[0106] The pharmaceutical composition may be formulated into a unit or multiple dosage form with pharma- ceutically acceptable carriers and / or additives by a method readily accessible to those skilled in the art. The formulation may be an oil or aqueous medium, a suspension, a syrup, an emulsion, an extract, a powder, a granule, a tablet or a capsule, and may further include a dispersant or stabilizer.

[0107] [Advantageous Effects of the Invention] The anti-HER2 / anti-4-1BB bispecific antibodies provided herein can activate 4-1BB signaling and enhance immune responses against high HER2-expressing cancers, as well as cancer cells with low HER2 expression and / or FcγRI expression characteristics. Thus, the bispecific antibodies may be useful as cancer immunotherapeutic agents that exhibit anti-cancer activity against cancers characterized by low HER2 expression and / or FcγRI expression. [Brief description of the drawings]

[0108] [Figure 1a] 1 is a graph showing the antigen (human 4-1BB) binding activity of anti-4-1BB antibodies measured by ELISA. [Figure 1b] 1 is a graph showing the binding activity of anti-4-1BB antibodies to 4-1BB-expressing cells, as measured by FACS. [Figure 2a] 1 is a graph showing the antigen (human HER2) binding activity of anti-HER2 / anti-4-1BB bispecific antibodies measured by ELISA. [Figure 2b] 1 is a graph showing the antigen (human HER2) binding activity of anti-HER2 / anti-4-1BB bispecific antibodies measured by ELISA. [Figure 3a] 1 is a graph showing the antigen (human 4-1BB) binding activity of an anti-HER2 / anti-4-1BB bispecific antibody, measured by ELISA. [Figure 3b] 1 is a graph showing the antigen (human 4-1BB) binding activity of an anti-HER2 / anti-4-1BB bispecific antibody, measured by ELISA. [Figure 4] FIG. 1 is a graph showing the efficacy of anti-HER2 / anti-4-1BB bispecific antibodies in inducing 4-1BB activation in FcγRI-expressing cells. [Figure 5a] FIG. 1 is a graph showing the effect of anti-HER2 / anti-4-1BB bispecific antibody on antibody-dependent cellular cytotoxicity (ADCC) in HER2 low-expressing tumor cells (JIMT-1). [Figure 5b] FIG. 1 is a graph showing the effect of anti-HER2 / anti-4-1BB bispecific antibody on antibody-dependent cellular cytotoxicity (ADCC) in HER2 low-expressing tumor cells (MDA-MB-231). [Figure 6] 1 is a graph showing the anti-cancer effect of an anti-HER2 / anti-4-1BB bispecific antibody against HER2 low-expressing tumors. [Figure 7] 1 is a graph showing the anti-cancer effect of simultaneous administration of an anti-HER2 / anti-4-1BB bispecific antibody and a PD-1 inhibitor against HER2 low-expressing tumors. EXAMPLES

[0109] The present invention will now be described in detail with reference to the following examples.

[0110] The following embodiments are intended only to illustrate the present invention and should not be construed as limiting the present invention.

[0111] Example 1. Anti-4-1BB antibody 1.1 Preparation of fully human monoclonal antibodies against 4-1BB Fully human monoclonal anti-4-1BB antibodies in full length IgG format were screened by immunotube panning of a phage library (obtained from KBio Health) against 4-1BB. For panning of the phage library against the target molecule, a total of four rounds of panning were performed using immunotubes coated with 4-1BB (NCBI accession number NP_001552.2).

[0112] Bacterial colonies from triplicate pannings were grown in 96-deep-well plates with SB-carbenicillin (Biomatik cat#A2311-5g) until they became cloudy, and 10 11 pfu of VCSM13 helper phage (K-Bio Health) was added. After infection for 1 h at 37° C. with gentle shaking (80 rpm), 70 μg / mL kanamycin was added and the cells were incubated overnight at 30° C. with shaking at 200 rpm.

[0113] The next day, the plates were centrifuged and the phage-containing supernatant was added to 4-1BB antigen-coated ELISA plates blocked with 3% (v / v) BSA (bovine serum albumin) in PBST (phosphate buffered saline with Tween 20). After 1 hour incubation at room temperature, the plates were washed three times with PBST and then anti-M13 antibody (Sino Biological cat#11973-MM05) was added. The plates were incubated for 1 hour, washed three times with PBST and the binding activity was measured using tetramethylbenzidine (TMB).

[0114] 4-1BB-specific binders were amplified for plasmid DNA sequencing. The light and heavy chain variable region (VL and VH) sequences were analyzed to identify unique sequences and determine sequence diversity as shown in Tables 6 to 13 (underlined: CDR1, CDR2, and CDR3, respectively). [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

[0115] 1.2. Preparation of scFv antibody against 4-1BB An anti-4-1BB scFv antibody having the structure of (N')-VL-linker-VH-(C') was prepared using the variable regions of fully human monoclonal antibodies against 4-1BB shown in Tables 6 to 13 in Example 1.1. The 44th amino acid residue "G" in the heavy chain variable region is substituted with "C", and the 103rd amino acid residue "G" in the light chain variable region is substituted with "C". These amino acid substitutions from "G" to "C" in the scFv may contribute to improved stability of bispecific antibodies that contain scFv as one target-specific moiety. The amino acid sequences of the prepared anti-4-1BB scFvs are shown in Tables 14 to 19 below, although one of skill in the art will recognize that in the following embodiments, the amino acid sequences can be altered or modified for specific purposes, including the application of various types of peptide linkers such as (GGGGS)2 (SEQ ID NO: 90), (GGGGS)3 (SEQ ID NO: 85), (GGGGS)4 (SEQ ID NO: 87), or (GS)9 (SEQ ID NO: 86). [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19]

[0116] 1.3. Antigen-binding ability of anti-4-1BB antibody (full-length IgG type) to human 4-1BB (1) Measurement of antigen-binding activity by ELISA To evaluate antigen-binding activity, the antibody candidates prepared in Example 1.1 were tested by ELISA. Briefly, microtiter plates were coated with 0.1 μg / ml human 4-1BB-Fc protein (Sino Biological) in PBS, incubated overnight at 4° C. with 100 μl / well, and blocked with 100 μl / well 5% (v / v) BSA.

[0117] Five-fold dilutions of humanized antibodies (1A10, 1A12, and AB41) starting at 10 μg / ml were added to each well and incubated for 1 to 2 hours at room temperature (RT). Plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Thermo) for 1 hour at room temperature. After washing, plates were developed with TMB substrate and analyzed spectrophotometrically at OD450-630 nm.

[0118] As a result, as shown in FIG. 1a, all of the anti-4-1BB antibodies tested exhibited 4-1BB binding activity.

[0119] (2) Cell-binding activity measured by FACS To assess cell binding activity, antibody candidates were analyzed for binding to human 4-1BB by fluorescence-activated cell sorting (FACS). TM NFκB-luc2 / 4-1BB Jurkat cell line (Promega; 3 × 10 5 Cells) were incubated with antibodies (1A10 and 1A12, 10 μg / mL each). After washing with FACS buffer (1% (v / v) BSA in PBS), FITC anti-human IgG antibody (Sigma, F9512, concentration: 2.0 mg / ml) was added to each well and incubated for 1 h at 4° C. The mean fluorescence intensity (MFI) of FITC was measured by FACSCalibur (BD Biosciences).

[0120] As a result, as shown in FIG. 1b, all of the tested anti-4-1BB antibodies exhibited efficient binding activity to 4-1BB expressed on the surface of human cells.

[0121] Example 2. Preparation of anti-HER2 antibody As the HER2-targeting portion of the anti-HER2 / anti-4-1BB bispecific antibody, trastuzumab (Genentech; hereafter "HER2(WT)", DrugBank accession number DB00072; human IgG1 kappa monoclonal antibody) or an antigen-binding fragment thereof, such as scFv, was used.

[0122] The sequence of HER2(WT) is summarized in Table 20 below. The constant region of the anti-HER2 antibody contained in the bispecific antibody can be modified by introducing one or more mutations or changes into human IgG1. As an exemplary embodiment, HER2 (NA or N297A) is shown in Table 20 below. [Table 20]

[0123] Example 3. Preparation of anti-HER2 / anti-4-1BB bispecific antibody Various anti-HER2 / anti-4-1BB bispecific antibody candidates were prepared in either full-length IgG (anti-HER2 antibody)-scFv (anti-4-1BB antibody) or full-length IgG (anti-4-1BB antibody)-scFv (anti-HER2 antibody) formats. In this embodiment, the anti-HER2 IgG and 4-1BB scFv clones prepared in Example 2 and Example 1.2, respectively, were exemplarily selected to prepare anti-HER2 / anti-4-1BB bispecific antibodies in the form of IgG-scFv fusions (wherein the scFv antibody fragment of one antigen is fused to the C-terminus of the IgG of the other antigen). IgG1 with a mutant backbone with reduced ADCC (such as the N297A mutation, Cancer Cell, vol. 19, issue 1, pp. 101-113) was used when the HER2 targeting moiety was located in the full IgG moiety, and IgG4 was used when the 4-1BB targeting moiety was located in the full IgG moiety.

[0124] A DNA segment 1 having a nucleotide sequence encoding the heavy chain of the IgG antibody of the anti-HER2 / anti-4-1BB bispecific antibody was inserted into pcDNA3.4 (Invitrogen, A14697; plasmid 1), and a DNA segment 2 having a nucleotide sequence encoding the light chain of the IgG antibody of the anti-HER2 / anti-4-1BB bispecific antibody was inserted into pcDNA3.4 (Invitrogen, A14697; plasmid 2). Next, a vector for expressing the bispecific antibody was prepared by fusing the DNA segment 3 encoding the scFv to a portion of the DNA segment 1 corresponding to the C-terminus of the Fc region of the IgG antibody inserted into the plasmid 1 using a DNA segment 4 encoding a 15-amino acid-long peptide linker consisting of (GGGGS)3 (SEQ ID NO: 85) or a DNA segment 5 encoding an 18-amino acid-long peptide linker consisting of (GS)9 (SEQ ID NO: 86). Furthermore, to stabilize the scFv, further modifications were applied to create disulfide bridges fusing VL103-VH44 (VL103: VL with a G→C mutation at position 103; VH44: VH with a G→C mutation at position 44) to the C-terminus of the light chain and the C-terminus of the heavy chain, respectively, as described in Example 1.2.

[0125] The sequences of the heavy chains, light chains, scFvs, and DNA fragments used to generate some exemplary bispecific antibodies are illustrated in Tables 21 to 29. The antibodies shown below may have one or more point mutations introduced into the amino acid sequence to improve stability and potency, reduce immunogenicity, etc. [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29]

[0126] Example 4. Testing the binding affinity of bispecific antibodies (BsAbs) 4.1. Binding to human HER2 HER2 binding affinity measurement of the bispecific antibodies was carried out by ELISA with reference to Example 1.3(1). Briefly, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with 100 μl / well of 1 μg / ml human HER2-His protein (Sino Biological, 10001-H08B) in PBS overnight at 4° C., and then blocked with blocking buffer (200 μl / well of 1% BSA in PBS, containing bovine serum albumin (Gibco, 30063572)) at 37° C. for 2 hours. Serial dilutions (starting from 0.1 μM) of the anti-HER2 / anti-4-1BB bispecific antibody prepared in Example 3 and the anti-HER2 antibody (HER2(NA)) as a control were added to each well and incubated at 37° C. for 1 hour. The plate was washed with PBS / 0.05% Tween20 and incubated with HRP-conjugated Fab antibody (Pierce, 31414) at 37° C. for 1 hour. After washing, plates were developed with tetramethylbenzidine (TMB, Sigma, T0440) substrate and analyzed spectrophotometrically at OD 450-650 nm.

[0127] As shown in Figures 2a and 2b, the results confirmed that all tested anti-HER2 / anti-4-1BB bispecific antibodies were able to bind to human HER2 protein with high affinity similar to the control anti-HER2 antibody (NA).

[0128] 4.2. Binding affinity: ELISA measurement for human 4-1BB The binding affinity measurement of the bispecific antibody to 4-1BB was carried out by ELISA with reference to Example 1.3(1). Briefly, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with 1 μg / ml human 4-1BB-His protein (Sino Biological, 10041-H08H) in PBS at 100 μl / well overnight at 4° C. The plates were blocked with blocking buffer (1% (v / v) bovine serum albumin (BSA) (Gibco, 30063572) in PBS at 200 μl / well) at 37° C. for 2 hours. Serial dilutions (starting from 0.1 μM) of the anti-HER2 / anti-4-1BB bispecific antibody prepared in Example 3 and the anti-HER2 antibody (HER2(NA)) as a control were added to each well and incubated at 37° C. for 1 hour. Plates were washed with PBS / 0.05% Tween 20 and incubated with HRP-conjugated Fab antibody (Pierce, 31414) for 1 hour at 37° C. After washing, plates were developed with TMB substrate and analyzed spectrophotometrically at OD 450-650 nm.

[0129] As shown in Figures 3a and 3b, all tested anti-HER2 / anti-4-1BB bispecific antibodies were able to bind to human 4-1BB protein with high affinity, whereas the anti-HER2 antibody did not bind to human 4-1BB protein.

[0130] The results of Figures 2a, 2b, 3a, and 3b are quantified and summarized in Table 30 below. [Table 30]

[0131] As shown in Table 30, all tested anti-HER2 / anti-4-1BB bispecific antibodies were able to bind with high affinity to both human HER2 and human 4-1BB proteins.

[0132] 4.3. Binding to various human HER2-expressing cell surfaces See Example 1.3(2) above, binding affinity measurements of bispecific antibodies to the surface of various cells expressing HER2 were carried out by FACS analysis.

[0133] Various tumor cell lines were used, as listed in Table 31 below. After each cell line was isolated and washed with PBS, cells were counted and cultured at 2×10 5 The cells were set at 100 μl cells / 100 μl FACS buffer, then treated with 10 μg / mL anti-HER2 antibody or anti-HER2 / anti-4-1BB bispecific antibody, and reacted for 1 hour at 4° C. After the reaction, the cells were washed with FACS buffer, incubated with FITC-labeled constant region (Fc)-specific antibody (goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 2.0 mg / ml), and then incubated at 2 μl / 2×10 5 After suspending the cells in 100μl FACS buffer, the cells were reacted at 4℃ for 1 hour. After the reaction, the cells were washed with FACS buffer and analyzed using a FACSCalibur instrument. The negative control was treated with FITC-labeled constant region (Fc) specific antibody alone. To compare the expression level of HER2 among cancer cell lines, the peak shift result value of the experimental group was divided by the peak shift result value of the negative control group.

[0134] The results obtained are shown in Table 31 below. [Table 31]

[0135] As shown in Table 31, all tested anti-HER2 / anti-4-1BB bispecific antibodies are capable of binding to the cell surface expressing human HER2 protein.

[0136] 4.4. Binding affinity to 4-1BB_SPR measurement In the SPR experiments, anti-HER2 / anti-4-1BB bispecific antibodies were captured separately on flow cells 2, 3, and 4, and flow cell 1 was kept as a reference. Anti-human Fab antibodies (GE Healthcare, 28958325) were immobilized on a Biocore® series S sensor chip CM5 (GE Healthcare, BR100530) by amine coupling. Recombinant human 4-1BB protein (ACROBiosystems, 41B-H5227) was flowed over the chip at 30 μl / min for 300 s at concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 nM, respectively, followed by a dissociation step of 400 s. Regeneration was performed using 10 mM glycine-HCl (pH 2.0) (GE Healthcare, BR100355).

[0137] The results obtained are shown in Table 32 below. [Table 32]

[0138] As shown in Table 32, the anti-HER2 / anti-4-1BB bispecific antibodies tested exhibited high 4-1BB binding affinity.

[0139] Example 5. HER2 expression levels in various cells HER2 cell surface expression levels in various cancer cell lines were quantified using a QIFIKIT quantification kit (Dako) according to the manufacturer's recommendations. More specifically, cells were stained with unlabeled anti-HER2 mouse monoclonal antibody (R&D Systems) or purified mouse IgG2b isotype control (R&D Systems) at saturating concentrations. After washing, the stained cells and calibration beads in the kit were simultaneously labeled with the same FITC-conjugated goat anti-mouse IgG secondary antibody in the kit. The labeled cells and calibration beads were analyzed by flow cytometer. Linear regression was performed using the MFI values ​​of the calibration beads. Antibody binding capacity (ABC) was determined by extrapolation from this regression line, and specific ABC (sABC) was determined by subtracting the ABC of the isotype control antibody from the ABC of the anti-HER2 antibody. Furthermore, the sABC (HER2 levels) of various cell lines obtained above were normalized to the value of SK-BR-3 (100%).

[0140] The results obtained are shown in Table 33. [Table 33]

[0141] Example 6. Induction of 4-1BB signal activation dependent on FcγRI binding of anti-HER2 / anti-4-1BB bispecific antibody CHO-K1 cells (GenScript) expressing FcγRI (CD64) were plated in 96-well assay plates (4 × 10 4 cells / well) and incubated overnight at 37°C in a 5% CO2 incubator. On the day of the assay, the culture medium was removed from each well, and effector cells (NFκB-Luc2 / h4-1BB cells; Promega) were plated (5 × 10 4 Each well was treated with anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)×1A10 M12) or anti-4-1BB antibody (urelumab, U.S. Pat. No. 7,288,638) and incubated in an incubator at 37° C. and 5% CO2 for 6 hours. Then, Bio-Glo TMReagent was added to each well and luminescence was measured using a microplate reader.

[0142] As a result, as shown in FIG. 4, the anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)×1A10 M12) induced strong 4-1BB activation in the presence of FcγRI.

[0143] Example 7. FcγRIIIa-mediated ADCC activity of anti-HER2 / anti-4-1BB bispecific antibodies in HER2-low expressing tumor cells To determine the efficacy of the anti-HER2 / anti-4-1BB bispecific antibody in HER2-low expressing tumor cells, the effect on FcγRIIIa-mediated antibody-dependent cellular cytotoxicity (ADCC) was examined. HER2-low expressing cells JIMT-1 and MDA-MB-231 (see Table 33) were plated in 96-well assay plates (1×10 4 cells / well) and incubated overnight at 37°C in a 5% CO2 incubator. On the day of the assay, the culture medium was removed from each well, and effector cells (NFκB-Luc2 / FcγRIIIa cells; Promega) were plated (5 × 10 4 Each well was treated with anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)×1A10 M12), US'250Ab (anti-HER2 / anti-4-1BB bispecific antibody, prepared according to SEQ ID NOs: 9 and 10 in U.S. Pat. No. 10,865,250), or anti-4-1BB antibody, urelumab, and incubated at 37° C., 5% CO2 in an incubator for 6 hours. Then, Bio-Glo TM Reagent was added to each well and luminescence was measured using a microplate reader.

[0144] As a result, as shown in Figures 5a (JIMT-1 cell line) and 5b (MDA-MB-231 cell line), in both of the HER2 low-expressing tumor cells tested (JIMT-1, MDA-MB-231), the anti-HER2 / anti-4-1BB bispecific antibody US'250Ab and the anti-4-1BB antibody urelumab hardly induced ADCC activation, whereas the anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT) x 1A10 M12) significantly induced ADCC activation.

[0145] Example 8. Antitumor effect of anti-HER2 / anti-4-1BB bispecific antibodies in a mouse model bearing HER2 low-expressing tumors A mouse model was prepared to evaluate the efficacy of the anti-HER2 / anti-4-1BB bispecific antibody against HER2-low expressing tumors. Specifically, mouse colon cancer cells MC38 were genetically engineered to express human HER2 (MC38 / hHER2, Biocytogen) and wild-type MC38 tumor cells were co-cultured at a 3:7 ratio based on cell number (MC38 / hHER2 1.5 × 10 6 Cells + MC38 3.5×10 6 cells, 0.1 mL PBS) or a 1:9 ratio (MC38 / hHER2 0.5 × 10 6 Cells + MC38 4.5×10 6 The cells were combined in 0.1 mL PBS to prepare tumor cells that mimicked the environment of low human HER2 expression. The tumor cells were implanted into the flanks of mice genetically engineered to express human 4-1BB (h4-1BB) (h4-1BB knock-in mice, Biocytogen), and the mice were randomized based on tumor volume on the fourth day after implantation.

[0146] Mice were grouped to have comparable tumor volumes and intraperitoneally administered anti-hIgG1 antibody, US'250Ab, and anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)x1A10 M12) at doses of 2.25 mg / kg, 3 mg / kg, and 3 mg / kg on the day of grouping, 4 days later, and 7 days later, respectively, and tumor volumes were then measured twice weekly with digital calipers. As shown in Figure 6, anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)x1A10 M12) showed superior tumor inhibition in both tumor mouse models mimicking HER2 low-expressing tumors (3:7 ratio model and 1:9 ratio model) compared to another anti-HER2 / anti-4-1BB bispecific antibody US'250Ab and anti-hIgG1 antibody.

[0147] Example 9. Antitumor effect of co-administration of anti-HER2 / anti-4-1BB bispecific antibody and PD-1 inhibitor in a mouse model bearing HER2 low-expressing tumors A mouse model was constructed to evaluate the antitumor effect of co-administration of an anti-HER2 / anti-4-1BB bispecific antibody and a PD-1 inhibitor (anti-PD-1 antibody) against HER2-low expressing tumors. Specifically, mouse colon cancer cells MC38 genetically engineered to express human HER2 (MC38 / hHER2; Biocytogen) and wild-type MC38 tumor cells were co-administered at a cell number ratio of 1:9 (MC38 / hHER2 0.5 × 10 6 Cells + MC38 4.5×10 6 The cells were mixed with 0.1 mL PBS (containing 0.1 mL of HER2 cells) to prepare tumor cells that mimicked the environment of low human HER2 expression. The tumor cells prepared above were implanted into the flanks of mice genetically engineered to express human 4-1BB (h4-1BB) (h4-1BB knock-in mice; Biocytogen), and the mice were randomized based on tumor volume 6 days after implantation.

[0148] Mice were grouped so that the tumor volumes were equivalent, and each group received Anti-hIgG1 (2.25 mg / kg, BIW for 2 weeks) + anti-mIgG2 antibody (10 mg / kg, QW for 4 weeks) (isotype control group), Anti-mPD-1 antibody (BioXcell, Cat#BP0146; 10mg / kg, QW 4 weeks), Anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)×1A10 M12; 3 mg / kg, BIW for 2 weeks), and Anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)×1A10 M12; 3mg / kg, BIW 2 weeks) + anti-mPD-1 antibody (10mg / kg, QW 4 weeks) was administered intraperitoneally, and tumor volumes were measured twice weekly with digital calipers.

[0149] As shown in FIG. 7, the anti-HER2 / anti-4-1BB bispecific antibody (HER2(WT)×1A10 M12) not only exhibited excellent anti-tumor effects when administered alone, but also exhibited enhanced anti-tumor effects against tumors mimicking a low HER2-expressing environment when co-administered with the anti-mPD-1 antibody, compared with each agent alone.

[0150] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference in its entirety.

[0151] The use of the terms "a" and "an" and "the" and "at least one" and "more than one" and similar references in the context of describing the invention (particularly in the context of the claims which follow) are to be construed as including both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The use of the term "at least one" (or "one or more") followed by a list of one or more items (e.g., "at least one of A and B") is to be construed as meaning one selected item. Unless otherwise specified herein or clearly contradicted by context, the terms "comprising," "having," "including," and "containing," as used herein, as referring to a listed item (A or B) or any combination of two or more of the listed items (A and B), are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise specified herein or clearly contradicted by context. References to ranges of values ​​in this specification are intended merely as a shorthand way of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually set forth herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of examples or exemplary language (e.g., "etc.") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise stated in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0152] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that such modifications may occur to those of skill in the art, and it is intended that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this invention includes any combination of the above-described elements in all possible variations unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A pharmaceutical composition for the prevention or treatment of HER2-low expression cancer comprising an anti-4-1BB / anti-HER2 bispecific antibody, wherein the bispecific antibody is (a) with an anti-4-1BB antibody or its antigen-binding fragment; (b) Anti-HER2 antibody or its antigen-binding fragment It includes an anti-4-1BB antibody or its antigen-binding fragment, Sequence ID: H-CDR1 containing amino acid sequence 1, 2, or 3; Sequence ID: H-CDR2 containing amino acid sequence 4, 5, or 6; Sequence ID: H-CDR3 containing amino acid sequence 7, 8, 9, 10, or 11; Sequence ID: L-CDR1 containing amino acid sequence 12 or 13; Sequence ID: L-CDR2 containing amino acid sequence 14 or 15; and Sequence ID: L-CDR3 containing amino acid sequence 16 or 17 A pharmaceutical composition containing [the specified substance].

2. Anti-4-1BB antibody or its antigen-binding fragment, The pharmaceutical composition according to claim 1, comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 1; H-CDR2 containing the amino acid sequence of SEQ ID NO: 4; H-CDR3 containing the amino acid sequence of SEQ ID NO: 8; L-CDR1 containing the amino acid sequence of SEQ ID NO: 12; L-CDR2 containing the amino acid sequence of SEQ ID NO: 14; and L-CDR3 containing the amino acid sequence of SEQ ID NO:

16.

3. Anti-4-1BB antibody or its antigen-binding fragment, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29; Light chain variable region containing amino acid sequence 30, 31, 32, 33, 34, or 88. A pharmaceutical composition according to claim 1, comprising:

4. Anti-4-1BB antibody or its antigen-binding fragment, (i) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 34; or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and light chain variable region containing the amino acid sequence of SEQ ID NO: 31 A pharmaceutical composition according to claim 1, comprising:

5. Anti-4-1BB antibody or its antigen-binding fragment, A heavy chain containing the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, 60, or 61; Light chain containing amino acid sequence number 62, 63, or 64 A pharmaceutical composition according to claim 1, comprising:

6. The pharmaceutical composition according to claim 1, wherein the anti-4-1BB antibody or its antigen-binding fragment is the anti-4-1BB scFv of the anti-4-1BB antibody.

7. The pharmaceutical composition according to claim 1, wherein the anti-HER2 antibody is trastuzumab, pertuzumab, or trastuzumab emtansine (T-DM1).

8. An anti-HER2 antibody or its antigen-binding fragment, Sequence ID: H-CDR1 containing amino acid sequence 65; Sequence ID: H-CDR2 containing amino acid sequence 66; Sequence ID: H-CDR3 containing amino acid sequence 67; Sequence ID: L-CDR1 containing 68 amino acid sequences; Sequence ID: L-CDR2 containing the amino acid sequence of 69; and Sequence ID: L-CDR3 containing amino acid sequence 70 A pharmaceutical composition according to claim 1, comprising:

9. An anti-HER2 antibody or its antigen-binding fragment, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 71; SEQ ID NO: 72 Light chain variable region containing amino acid sequence A pharmaceutical composition according to claim 1, comprising:

10. An anti-HER2 antibody or its antigen-binding fragment, SEQ ID NO: A heavy chain containing the amino acid sequence of 73 or 74; Sequence ID: Light chain containing amino acid sequence 75 A pharmaceutical composition according to claim 1, comprising:

11. The pharmaceutical composition according to claim 1, wherein the anti-HER2 antibody or its antigen-binding fragment is the anti-HER2 scFv of the anti-HER2 antibody.

12. Anti-4-1BB / anti-HER2 bispecific antibody, (a) The full-length form of the anti-HER2 antibody and the scFv of the anti-4-1BB antibody, or (b) Full-length form of anti-4-1BB antibody and scFv of anti-HER2 antibody A pharmaceutical composition according to claim 1, comprising:

13. Anti-4-1BB / anti-HER2 bispecific antibody, Sequence ID: 83; the first polypeptide containing the amino acid sequence; Sequence ID: Second polypeptide containing amino acid sequence 75 A pharmaceutical composition according to claim 1, comprising:

14. The pharmaceutical composition according to claim 1, further comprising a cancer immunotherapy agent.

15. The pharmaceutical composition according to claim 14, wherein the cancer immunotherapy agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a combination thereof.

16. A pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer has an immunohistochemistry (IHC) score of 0, 1+, or 2+ when measured by IHC using an anti-HER2 antibody specific to HER2.

17. The pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer has 50% or less HER2-expressing tumor cells based on all tumor cells.

18. The pharmaceutical composition according to any one of claims 1 to 15, wherein, when measured by a general protein assay method, the tumor has a lower HER2 level than that in the HCC1954 cell line.

19. The pharmaceutical composition according to any one of claims 1 to 15, wherein, when measured by a general protein assay method, the tumor has an intratumoral HER2 level of 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less compared to the HER2 level in the SK-BR-3 cell line.

20. The pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer is characterized by FcγRI expression.

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