Anti-B7-H4 / anti-4-1BB bispecific antibody and its applications

Abstract

The present invention provides an anti-B7-H4 / anti-4-1BB bispecific antibody that can effectively block the T-cell suppressive effect of B7-H4 on tumor cell surface and activate 4-1BB on T-cell. The bispecific antibody can have high binding affinity to both B7-H4 protein (e.g., human, monkey and mouse B7-H4 protein) and 4-1BB protein (e.g., human, monkey and mouse 4-1BB protein). The present disclosure further provides an anti-B7-H4 antibody that specifically binds to B7-H4 protein.

Description

[Technical Field]

[0001] This invention relates to anti-B7-H4 antibodies, anti-B7-H4 / anti-4-1BB bispecific antibodies, and their applications. [Background technology]

[0002] B7-H4 is a single-pass type I transmembrane protein and a member of the B7 superfamily of proteins that provide co-signaling along with T-cell receptor antigen signaling. B7-H4 is a negative regulator of T cells, and ligation of T cells with B7-H4 suppresses their growth, cytokine secretion, and cytotoxicity.

[0003] Human B7-H4 is a 282-amino acid protein (including an amino-terminal signaling sequence), of which 235 amino acids are predicted to be in extracellular space after cleavage of the amino-terminal signaling sequence. B7-H4 contains an Ig-like V-domain, an Ig-like C-domain, a transmembrane domain, and a short cytoplasmic tail.

[0004] B7-H4 has the potential to modulate the immune system downwards through co-inhibitory signaling of T cell receptor antigen-dependent signaling. While nominally expressed in normal human tissue, B7-H4 is highly overexpressed in a vast number of human cancers, including female reproductive system cancers—breast, ovarian, and endometrial cancers. The prevalence of B7-H4 has been reported to be high in invasive ductal carcinoma and lobular carcinoma, including both primary (-95%) and metastatic breast cancer (-97%). Increased B7-H4 staining was associated with negative PR and Her2 status, but expression was independent of tumor grade or stage. In these types of breast cancer, a high percentage of B7-H4 stained cells was accompanied by a decrease in invasive lymphocyte counts.

[0005] 4-1BB is a member of the TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule that is expressed after the activation of immune cells in both innate and adaptive immune cells. 4-1BB plays an important role in regulating the activities of various immune cells. The 4-1BB signaling pathway enhances the proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Various other studies have shown that the activation of 4-1BB enhances the immune response that eliminates tumors in mice. Therefore, it is suggested that 4-1BB is a promising target molecule in cancer immunology. Despite these anti-tumor efficacy, anti-4-1BB antibodies have induced severe hepatotoxicity in clinical applications.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide an anti-B7-H4 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof.

[0007] Another object of the present invention is to provide an anti-B7-H4 antibody or an antigen-binding fragment thereof.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases related to B7-H4, 4-1BB, or both of them.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating B7-H4-related diseases thereof.

[0010] Another object of the present invention is to provide a method for preventing or treating diseases related to B7-H4, 4-1BB, or both of them in an individual.

[0011] Another object of the present invention is to provide a method for preventing or treating a disease related to B7-H4 in an individual.

[0012] Another object of the present invention is to provide an anti-B7-H4 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof for use in preventing or treating a disease related to B7-H4, 4-1BB, or both.

[0013] Another object of the present invention is to provide an anti-B7-H4 antibody or an antigen-binding fragment thereof for use in preventing or treating a disease related to B7-H4.

Means for Solving the Problems

[0014] Definition

[0015] It should be noted that in the present invention, the term "one" ("a" or "an") entity refers to one or more of that entity. For example, "antibody" is understood to indicate one or more antibodies. Thus, the terms "one" ("a" or "an"), "one or more", and "at least one" can be used interchangeably in the present invention.

[0016] In the present invention, the terms "composed of a sequence", "composed of an essential sequence", or "including a sequence" can refer to any case including a sequence, but are not intended to exclude cases including additional sequences other than the said sequence.

[0017] In the present invention, the terms "protein or polypeptide containing or composed of an amino acid sequence identified by an SEQ ID NO:" and "gene or polypeptide containing or composed of a nucleic acid sequence identified by an SEQ ID NO:" refer to a protein (or polypeptide) or gene (or polynucleotide) that consists of an essential amino acid sequence or nucleic acid sequence, or has an amino acid sequence or nucleic acid sequence with 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 while maintaining intrinsic activity / or function.

[0018] In this invention, the term "antibody" can encompass a wide variety of polypeptides that are biochemically distinguishable. Those skilled in the art should understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), some of which have subcategories (e.g., γ1-γ4), and light chains are classified as kappa or lambda (Κ, λ). It is characteristic of these chains that the "classification" of antibodies is determined to be IgG, IgM, IgA, IgG, or IgE, respectively. Subclassifications (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and are known to provide functional specificity.

[0019] The term "heavy chain" may also refer to a full-length heavy chain or a fragment thereof, containing a variable region VH with an amino acid sequence sufficient to provide specificity to the antigen, and three invariant regions CH1, CH2, and CH3, and a hinge region. The term "light chain" refers to a full-length light chain or a fragment thereof, containing a variable region VL- with an amino acid sequence sufficient to provide specificity to the antigen and an invariant region CL. It can also refer to a fragment of this.

[0020] The term "complementarity-determining region (CDR)" refers to an amino acid sequence found in the hypervariable region of the heavy or light chain of an immunoglobulin. The heavy and light chains can each contain three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). CDRs can provide residues that play a crucial role in the antibody's binding to an antigen or epitope. The terms "specifically bind" or "specifically recognize" are well-known to those skilled in the art and indicate that an antibody and an antigen interact specifically to induce immunological activity.

[0021] In the present invention, antibodies may include, but are not limited to, polyclonal or monoclonal antibodies; and / or antibodies of human, humanized, animal (e.g., mouse, rabbit, etc.) origin; or chimeric antibodies (e.g., mouse-human chimeric antibodies). Furthermore, the antibodies of the present invention may include, but are not limited to, monospecific antibodies and multispecific antibodies such as bispecific and tripspecific antibodies.

[0022] In this invention, the term “antigen-binding fragment” refers to a fragment derived from a complete immunoglobulin structure that includes a portion capable of binding to an antigen, such as a CDR. For example, an antigen-binding fragment may be, but is not limited to, scFv, (scFv)2, Fab, Fab', F(ab')2, or any combination thereof. In this invention, an antigen-binding fragment may also be an antibody-derived fragment containing at least one complementarity-determining region selected from the group consisting of, for example, scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. For example, an antibody or antigen-binding fragment can be synthesized chemically or recombinantly (not spontaneously).

[0023] Anti-B7-H4 antibody

[0024] Anti-B7-H4 / anti-4-1BB bispecific antibodies may contain an anti-B7-H4 antibody or its antigen-binding fragment as a B7-H4 targeted moiety.

[0025] In one example, an anti-B7-H4 antibody or a fragment thereof can specifically bind to the B7-H4 protein (e.g., human B7-H4).

[0026] B7-H4 (also known as V-set Domain-Containing T Cell Activation Inhibitor 1 (VTCN1)), a member of the B7 family, is involved in the regulation of T cell activation. B7-H4 negatively regulates the T cell immune response by suppressing T cell proliferation cytokine secretion and the cell cycle. From a structural standpoint, B7-H4 is a member of the immunoglobulin (Ig) superfamily, containing two Ig-like domains: a variable (IgV) domain and an immutable (IgC) domain. Similar to the other negative costimulator PD-L1, B7-H4 is expressed not only in APCs (antigen-presenting cells) but also in a variety of ordinary non-lymphoid and cancerous organisms. Expression by non-APCs suggests that B7-H4 may have additional functions as one of the classical costimulatory molecules.

[0027] The human protein B7-H4 consists of the amino acid sequence of NCBI reference sequence ID: NP_001240778.1, NP_001240779.1, or NP_078902.2, and the nucleic acid sequence is NM_001253849.2, NM_001253850.2, or NM_024626.4. Unless otherwise evident in the context used in this invention, B7-H4 refers to human B7-H4, although the antibody also has the ability to bind to monkey B7-H4. The amino acid sequence of mouse B7-H4 is GenBank:XP_001103 It is represented as 715.

[0028] Overexpression of B7-H4 with organizational specificity has correlated with negative clinical outcomes in prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, and renal cancer. Recently, we demonstrated that presentation of damaged B7-H4 exhibits type 1 diabetes in both mouse models and human patients, linking this process to proteolytic efflux of cell surface B7-H4 in the APC and pancreatic islets. Ig therapy reduced the incidence of T1D, experimental autoimmune encephalomyelitis, and rheumatoid arthritis, highlighting its importance as a potential therapeutic target for both autoimmune and cancer.

[0029] Anti-B7-H4 antibodies or their antigen-binding fragments can exhibit potent binding activity to B7-H4 and can be repurposed for therapeutic and / or diagnostic applications.

[0030] In one embodiment, an anti-B7-H4 / anti-4-1BB bispecific antibody comprises an anti-B7-H4 antibody or its antigen-binding fragment and an anti-4-1BB antibody or its antigen-binding fragment, wherein the anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, Heavy chain complementarity determination region 1 (CDR-H1) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 25, and 33; CDR-H2 containing amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 26, and 34; CDR-H3 containing amino acid sequences selected from the group consisting of SEQ ID NOs: 4, 27, and 35;

[0031] Light chain complementarity determination region 1 (CDR-L1) containing amino acid sequences selected from the group consisting of Sequence IDs 7, 8, 29, and 37; CDR-L2 containing amino acid sequences selected from the group consisting of SEQ ID NOs: 9 and 30; and CDR-L3 containing amino acid sequences selected from the group consisting of SEQ ID NOs: 10, 31, and 38; The anti-4-1BB antibody or its antigen-binding fragment comprises a heavy chain variable region containing at least one amino acid sequence selected from the group consisting of sequences 39 to 43; and a light chain variable region containing at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 52 and 53. The amino acid sequences of the heavy chain CDRs of the anti-B7-H4 antibody or its antigen-binding fragment are listed in Table 1.

[0032] [Table 1]

[0033] The amino acid sequences of the heavy chain variable region of the anti-B7-H4 antibody or its antigen-binding fragment are listed in Table 2.

[0034] [Table 2]

[0035] The amino acid sequences of the light chain CDRs of the anti-B7-H4 antibody or its antigen-binding fragment are listed in Table 3.

[0036] [Table 3]

[0037] The amino acid sequences of the light chain variable regions of anti-B7-H4 antibodies or their antigen-binding fragments are listed in Table 4.

[0038] [Table 4]

[0039] In one example, an anti-B7-H4 antibody or its antigen-binding fragment can be designed by appropriately mixing and matching the CDRs listed in Tables 1 and 3 to maintain its affinity for B7-H4. For example, an anti-B7-H4 antibody or its antigen-binding fragment may include the following: (1) CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 7, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; (2) CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; (3) CDR-H1 of SEQ ID NO: 25, CDR-H2 of SEQ ID NO: 26, CDR-H3 of SEQ ID NO: 27, CDR-L1 of SEQ ID NO: 29, CDR-L2 of SEQ ID NO: 30, and CDR-L3 of SEQ ID NO: 31; or (4) CDR-H1 of sequence number 33, CDR-H2 of sequence number 34, CDR-H3 of sequence number 35, CDR-L1 of sequence number 37, CDR-L2 of sequence number 9, and CDR-L3 of sequence number 38.

[0040] In one example, an anti-B7-H4 antibody or its antigen-binding fragment includes the following: Heavy chain variable region containing an amino acid sequence selected from the group consisting of Sequence IDs 1, 22, 24, and 32; and Light chain containing amino acids selected from the group consisting of SEQ ID NOs: 5, 6, 23, 28, and 36. Variable region.

[0041] Examples of heavy chain and light chain variable regions of antibody or antigen-binding fragments are listed in Tables 2 and 4.

[0042] In other embodiments, the variable regions of the heavy and light chains disclosed in Tables 2 and 4 can be appropriately combined (mixed and matched) for the production of various forms of antibodies, for example, they can form single-chain antibodies such as ScFV, or domain antibodies, or full-length antibodies (e.g., IgG-type antibodies containing two heavy chains and two light chains).

[0043] For example, an anti-B7-H4 antibody or antigen-binding fragment may include the following: (1) Heavy chain variable region of sequence number 1 and light chain variable region of sequence number 5; (2) Heavy chain variable region of sequence number 1 and light chain variable region of sequence number 6; (3) Heavy chain variable region of Sequence ID: 22 and light chain variable region of Sequence ID: 23; (4) Heavy chain variable region of SEQ ID NO: 24 and light chain variable region of SEQ ID NO: 28; or (5) The heavy chain variable region of sequence number 32 and the light chain variable region of sequence number 36.

[0044] In the present invention, each heavy chain variable region and light chain variable region may be combined with various invariant regions of each heavy chain and light chain to form the heavy chain and light chain of the complete antibody.

[0045] Non-restrictive examples of anti-B7-H4 antibodies or their antigen-binding fragments include: A heavy chain variable region comprising an amino acid sequence selected from the group consisting of Sequence IDs 1, 22, 24, and 32, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the aforementioned amino acid sequences; and A light chain variable region comprising an amino acid sequence selected from the group consisting of Sequence IDs 5, 6, 23, 28, and 36, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the aforementioned amino acid sequences.

[0046] In one example, an anti-B7-H4 antibody or its antigen-binding fragment comprises: heavy chain framework 1 (H-FR1) containing the amino acid sequence of SEQ ID NO: 12; heavy chain framework 2 (H-FR2) containing the amino acid sequence of SEQ ID NO: 13; heavy chain framework 3 (H-FR3) containing the amino acid sequence of SEQ ID NO: 14; heavy chain framework 4 (H-FR4) containing the amino acid sequence of SEQ ID NO: 15; light chain framework 1 (L-FR1) containing the amino acid sequence of SEQ ID NO: 17; light chain framework 2 (L-FR2) containing the amino acid sequence of SEQ ID NO: 18; light chain framework 3 (L-FR3) containing the amino acid sequence of SEQ ID NO: 19; and light chain framework 4 (L-FR4) containing the amino acid sequence of SEQ ID NO: 20.

[0047] In one embodiment, if the anti-B7-H4 antibody or antigen-binding fragment is in the form of an scFv, the heavy chain variable region and the light chain variable region can be linked through a suitable peptide linker. For example, the peptide linker may be (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9.

[0048] For example, the binding affinity of the anti-B7-H4 antibody of the present invention or its antigen-binding fragment to human B7-H4 is such that the dissociation constant or KD is 1 × 10⁻⁶. -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, or 1 × 10 -10 It is M or less.

[0049] Anti-4-1BB antibody

[0050] The anti-B7-H4 / anti-4-1BB bispecific antibody may include an anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB-targeted moiety. WO2020 / 111913, which discloses an anti-4-1BB antibody, is included herein in its entirety for reference.

[0051] In one example, an anti-4-1BB antibody or a fragment thereof can specifically bind to a 4-1BB protein (e.g., human 4-1BB). For example, the human 4-1BB protein may be selected from, but is not limited to, the group of proteins represented by NCBI registry number NP_001552.2, etc.

[0052] Such anti-4-1BB antibodies or antigen-binding fragments thereof can enhance immune responses or treat tumors (cancer) in mammals. These anti-4-1BB antibodies or antigen-binding fragments are characterized by their ability to localize and / or activate only within the tumor microenvironment (TME), or by significantly reducing hepatotoxicity compared to existing anti-4-1BB antibodies while maintaining immune response enhancement and / or tumor treatment efficacy.

[0053] In this invention, the term "4-1BB" refers to CD137 or TNFRSF9 (member 9 of the TNF receptor 25 superfamily), which is a member of the TNF receptor superfamily (TNFRSF) and a co-stimulatory molecule expressed after the activation of both innate and acquired immune cells. As used in this invention, 4-1BB may originate from mammals, for example, Homo sapiens (human) (NCBI registry number NP_001552.2).

[0054] As described in this invention, the term "4-1BB" includes variants, isotypes, homologs, orthologues, and paralogs. For example, an antibody specific to the human 4-1BB protein may, in certain cases, cross-react with 4-1BB proteins from non-human species. In other embodiments, an antibody specific to the human 4-1BB protein may be perfectly specific to the human 4-1BB protein, exhibit cross-reactivity with species or other types, or cross-react with 4-1BB from certain other species, but not all of them (e.g., cross-reacts with monkey 4-1BB but not mouse 4-1BB). The term "human 4-1BB" refers to the complete amino acid sequence of human 4-1BB, e.g., the complete amino acid sequence of human 4-1BB having NCBI registry number NP_001552.2. The term "mouse 4-1BB" refers to the complete amino acid sequence of mouse 4-1BB, e.g., the complete amino acid sequence of mouse 4-1BB having NCBI registry number NP033430.1.

[0055] 4-1BB is also known in the field, for example, as CD137.

[0056] The human 4-1BB sequence of the present invention may differ from the human 4-1BB of NCBI registry number NP_001552.2, for example, by having a conserved mutation or a mutation in a non-conserved region, and the 4-1BB sequence of the present invention has substantially the same biological function as the human 4-1BB of NCBI registry number NP_001552.2.

[0057] In one example, an anti-4-1BB antibody or its antigen-binding fragment may include the following: CDR-H1 containing amino acid sequences selected from SEQ ID NOs: 44 and 45; CDR-H2 containing amino acid sequences selected from SEQ ID NOs: 46 and 47; CDR-H3 containing amino acid sequences selected from sequence numbers 48 to 51; CDR-L1 containing the amino acid sequence of SEQ ID NO: 54; CDR-L2 containing the amino acid sequence of SEQ ID NO: 55; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56.

[0058] The amino acid sequences of the heavy chain CDRs of the anti-4-1BB antibody or its antigen-binding fragment are listed in Table 5.

[0059] [Table 5]

[0060] The amino acid sequences of the heavy chain variable region of the anti-4-1BB antibody or its antigen-binding fragment are listed in Table 6.

[0061] [Table 6]

[0062] The amino acid sequences of the light chain CDRs of the anti-4-1BB antibody or its antigen-binding fragment are listed in Table 7.

[0063] [Table 7]

[0064] The amino acid sequences of the light chain variable regions of the anti-4-1BB antibody or its antigen-binding fragment are listed in Table 8.

[0065] [Table 8]

[0066] In one example, an anti-4-1BB antibody or its antigen-binding fragment can be designed by appropriately mixing and matching the CDRs listed in Tables 5 and 7 to maintain its affinity for 4-1BB. For example, an anti-4-1BB antibody or its antigen-binding fragment may include the following: (1) CDR-H1 with SEQ ID NO: 44, CDR-H2 with SEQ ID NO: 46, CDR-H3 with SEQ ID NO: 48, CDR-L1 with SEQ ID NO: 54, CDR-L2 with SEQ ID NO: 55, and CDR-L3 with SEQ ID NO: 56; (2) CDR-H1 with SEQ ID NO: 44, CDR-H2 with SEQ ID NO: 46, CDR-H3 with SEQ ID NO: 49, CDR-L1 with SEQ ID NO: 54, CDR-L2 with SEQ ID NO: 55, and CDR-L3 with SEQ ID NO: 56; (3) CDR-H1 with SEQ ID NO: 44, CDR-H2 with SEQ ID NO: 46, CDR-H3 with SEQ ID NO: 50, CDR-L1 with SEQ ID NO: 54, CDR-L2 with SEQ ID NO: 55, and CDR-L3 with SEQ ID NO: 56; (4) CDR-H1 of SEQ ID NO: 44, CDR-H2 of SEQ ID NO: 46, CDR-H3 of SEQ ID NO: 49, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 56; or (5) CDR-H1 with sequence number 45, CDR-H2 with sequence number 47, CDR-H3 with sequence number 51, CDR-L1 with sequence number 54, CDR-L2 with sequence number 55, and CDR-L3 with sequence number 56.

[0067] Examples of heavy chain and light chain variable regions of antibody or antigen-binding fragments are listed in Tables 6 and 8.

[0068] Non-restrictive examples of anti-4-1BB antibodies or their antigen-binding fragments may include: A heavy chain variable region comprising an amino acid sequence selected from the group consisting of Sequence ID:39 to 43 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence described above; and A light chain variable region comprising an amino acid sequence selected from Sequence IDs 52 and 53 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences described above.

[0069] In other embodiments, the variable regions of the heavy and light chains disclosed in Tables 6 and 8 can be appropriately combined (mixed and matched) for the production of various forms of antibodies, for example, these can form single-chain antibodies such as scFv, or domain antibodies, or full-length antibodies (e.g., IgG-type antibodies containing two heavy chains and two light chains).

[0070] For example, an anti-4-1BB antibody or antigen-binding fragment may include the following: (1) Heavy chain variable region of sequence number 39 and light chain variable region of sequence number 52; (2) Heavy chain variable region of sequence number 40 and light chain variable region of sequence number 52; (3) Heavy chain variable region of Sequence ID: 41 and light chain variable region of Sequence ID: 52; (4) Heavy chain variable region of SEQ ID NO: 40 and light chain variable region of SEQ ID NO: 53; (5) Heavy chain variable region of sequence number 41 and light chain variable region of sequence number 53; (6) Heavy chain variable region of Sequence ID: 42 and light chain variable region of Sequence ID: 52; (7) The heavy chain variable region of sequence number 43 and the light chain variable region of sequence number 53.

[0071] In another embodiment, the anti-4-1BB antibody or its antigen-binding fragment may be an scFv (single-chain variable fragment) containing the following: A heavy chain variable region consisting of CDR-H1 containing amino acid sequences selected from SEQ ID NOs: 44 and 45, CDR-H2 containing amino acid sequences selected from SEQ ID NOs: 46 and 47, and CDR-H3 containing amino acid sequences selected from SEQ ID NOs: 48 to 51; a light chain variable region consisting of CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56; and a peptide linker between the heavy chain variable region and the light chain variable region.

[0072] More specifically, the anti-4-1BB antibody or its antigen-binding fragment may be an scFv containing the following: A heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 43; a light chain variable region containing an amino acid sequence selected from SEQ ID NOs: 52 and 53; and a peptide linker between the heavy chain variable region and the light chain variable region.

[0073] For example, an anti-4-1BB antibody or a fragment thereof may be an scFv containing the following: an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 43, or at least 80%, at least 85%, at least 90%, and at least the aforementioned amino acid sequence. A heavy chain variable region containing an amino acid sequence having sequence identity of 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and A light chain variable region comprising an amino acid sequence selected from the group consisting of Sequence IDs 52 and 53, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the aforementioned amino acid sequence; and a peptide linker between the heavy chain variable region and the light chain variable region.

[0074] For example, an anti-4-1BB antibody or its antigen-binding fragment may be an scFv containing the following: (1) Heavy chain variable region of sequence number 39 and light chain variable region of sequence number 52; (2) Heavy chain variable region of sequence number 40 and light chain variable region of sequence number 52; (3) Heavy chain variable region of Sequence ID: 41 and light chain variable region of Sequence ID: 52; (4) Heavy chain variable region of SEQ ID NO: 40 and light chain variable region of SEQ ID NO: 53; (5) Heavy chain variable region of sequence number 41 and light chain variable region of sequence number 53; (6) Heavy chain variable region of Sequence ID: 42 and light chain variable region of Sequence ID: 52; (7) Heavy chain variable region of Sequence ID: 43 and light chain variable region of Sequence ID: 53; A peptide linker between the heavy chain variable region and the light chain variable region.

[0075] In the present invention, the anti-4-1BB scFv can contain heavy-chain variable regions and light-chain variable regions in any order. For example, the anti-4-1BB scFv can contain light-chain variable regions and heavy-chain variable regions in the order from the N-terminus to the C-terminus. Alternatively, the anti-4-1BB scFv can contain heavy-chain variable regions and light-chain variable regions in the order from the N-terminus to the C-terminus.

[0076] In one embodiment, the heavy-chain and light-chain variable regions of scFv can be linked through a suitable peptide linker. For example, the peptide linker may be (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9.

[0077] Each of the heavy chain variable regions and light chain variable regions disclosed in the present invention may be combined with various invariant regions of the heavy and light chains to form the heavy and light chains of a complete antibody, respectively.

[0078] Such anti-4-1BB antibodies can be used for therapeutic purposes, such as treating various types of cancer, and can also be used for diagnostic and prognostic purposes.

[0079] The antibodies of the present invention are characterized by certain functional features or properties of the antibodies. For example, the antibodies can specifically bind to human 4-1BB and can bind to 4-1BB from certain other species, such as monkeys, such as cynomolgus monkeys, rhesus monkeys, but may not substantially bind to 4-1BB from certain other species, such as mouse 4-1BB. Preferably, the antibodies disclosed in the present invention bind to human 4-1BB with high affinity.

[0080] The binding of the antibodies of the present invention to 4-1BB can be evaluated using one or more techniques well established in the art. For example, in a preferred embodiment, the antibodies are tested by flow cytometry by reacting with cell lines expressing human 4-1BB, such as 4-1BB, such as human 4-1BB or monkey 4-1BB, such as rhesus monkey or cynomolgus monkey, or CHO cells transfected to express mouse 4-1BB on the cell surface. Other cells suitable for use in flow cytometry include anti-CD3-stimulated CD4 + activated T cells containing intrinsic 4-1BB.

[0081] Also, other suitable binding assays include, for example, ELISA assays using recombinant 4-1BB protein. Additionally or alternatively, the binding of the antibodies, including binding kinetics (e.g., KD values), can be tested by Octet analysis. For example, the binding affinity of the anti-4-1BB antibodies of the present invention or antigen-binding fragments thereof to human 4-1BB is 1×10 -6 M or less, 1×10 -7 M or less, 1×10 <C -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less or those having a dissociation -10 constant) or KD of 1.80×10 M or less, etc.

[0082] The anti-4-1BB antibody or a fragment thereof, the anti-B7-H4 antibody or a fragment thereof, and / or the bispecific antibody may be, but is not limited to, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0083] In one example, the antibody or fragment thereof does not occur naturally or is synthesized chemically or recombinantly. Considering that each of these antibodies can bind to 4-1BB (e.g., human 4-1BB) and / or B7-H4 (e.g., human B7-H4), the CDR sequence or V H and V L The sequences may be “mixed and matched” to produce other anti-4-1BB binding molecules and / or other anti-B7-H4 binding molecules. Specifically, CDR sequences or V H and V L When chains are mixed and matched, for example, a certain V H / V L V from the pair H The array is structurally similar to V H The sequence may be replaced. Similarly, preferably a specific V H / V L V from the pair L The array is structurally similar to V L The array may be replaced.

[0084] Anti-B7-H4 / anti-4-1BB bispecific antibody

[0085] The bispecific antibody of the present invention may include the following: (1) an anti-B7-H4 antibody or an antigen-binding fragment thereof that can specifically recognize and / or bind to the B7-H4 protein; and (2) An anti-4-1BB antibody or an antigen-binding fragment thereof that can specifically recognize and / or bind to the 4-1BB protein.

[0086] The bispecific antibody of the present invention can bind to both B7-H4 and 4-1BB. The bispecific antibody, consisting of an anti-B7-H4 antibody or its antigen-binding fragment and an anti-4-1BB antibody or its antigen-binding fragment, is as described above.

[0087] Bispecific antibodies can be advantageous due to the function of anti-B7-H4 antibody or a fragment thereof and / or anti-4-1BB antibody or a fragment thereof.

[0088] In one embodiment, a bispecific antibody can have the ability to enhance cytokine secretion by containing an anti-B7-H4 antibody or its antigen-binding fragment. Furthermore, by containing an anti-4-1BB antibody or its antigen-binding fragment, the bispecific antibody of the present invention can bind to human 4-1BB and exhibit the ability to activate T cells. The anti-B7-H4 / anti-4-1BB bispecific antibody can activate 4-1BB signaling under conditions of B7-H4-expressing tumor cells. Moreover, the anti-4-1BB antibody or its antigen-binding fragment contained in the bispecific antibody is characterized by localizing and / or activating / / or having significantly reduced hepatotoxicity compared to before, only in the tumor microenvironment (TME).

[0089] Furthermore, anti-4-1BB antibodies enhance the immune response and / or maintain the efficacy of tumor treatment.

[0090] Methods for evaluating the ability of antibodies to stimulate an immune response include, for example, in vivo tumor transplantation models, This includes evaluating its ability to suppress tumor growth.

[0091] A bispecific antibody comprising a B7-H4 targeted moisture and a 4-1BB targeted moisture, wherein one of the B7-H4 targeted moisture and the 4-1BB targeted moisture may be a full-length antibody, and the other may be, but not limited to, an antigen-binding fragment (e.g., scFv) containing a heavy chain CDR, a light chain CDR, or a combination thereof.

[0092] Full-length antibodies targeting one of the B7-H4 and 4-1BB proteins and antigen-binding fragments targeting the remaining proteins may be linked directly or chemically (e.g., covalently) via a peptide linker. The antigen-binding fragment (e.g., scFv) can be linked directly or via a peptide linker to the N-terminus of the full-length antibody (e.g., the N-terminus of the light or heavy chain of the full-length antibody), the C-terminus of the full-length antibody (e.g., the C-terminus of the heavy chain (or Fc or CH3 domain) of the full-length antibody), or both of these.

[0093] In one embodiment, a bispecific antibody may include a full-length anti-B7-H4 antibody, an antigen-binding fragment (e.g., scFv) of an anti-4-1BB antibody, and a peptide linker between them. In another embodiment, a bispecific antibody may include a full-length anti-4-1BB antibody, an antigen-binding fragment (e.g., scFv) of an anti-B7-H4 antibody, and a peptide linker between them.

[0094] In one embodiment, an scFv contained in a bispecific antibody can contain heavy chain variable regions and light chain variable regions in any order. For example, an scFv contained in a bispecific antibody may contain heavy chain variable regions and light chain variable regions in the N-terminus to C-terminus direction, and selectively contain peptide linkers between them, or alternatively, an scFv contained in a bispecific antibody may contain light chain variable regions and heavy chain variable regions in the N-terminus to C-terminus direction, and selectively contain peptide linkers between them.

[0095] In one example, an anti-B7-H4 / anti-4-1BB bispecific antibody activates 4-1BB signaling, which in turn activates an immune response via B7-H4 expressed on the cell surface.

[0096] If the bispecific antibody includes a full-length anti-B7-H4 antibody and an anti-4-1BB scFv, the bispecific antibody may include the following: (i) First polypeptide from N-terminus to C-terminus: Heavy chain of anti-B7-H4 antibody, Selectively, peptide linkers (first peptide linkers), and Anti-4-1BB scFv; and (ii) Second polypeptide containing the light chain of anti-B7-H4 antibody, The aforementioned anti-4-1BB scFv is oriented from the N-terminus to the C-terminus. The light chain variable region of the anti-4-1BB antibody, Selectively, peptide linkers (second peptide linkers), and The heavy chain variable region of the anti--4-1BB antibody.

[0097] Furthermore, selectively, bispecific antibodies may include the following: (i) First polypeptide from N-terminus to C-terminus: Heavy chain of anti-B7-H4 antibody, Selectively, peptide linkers (first peptide linkers), and Anti-4-1BB scFv; and (ii) Second polypeptide containing the light chain of anti-B7-H4 antibody, The aforementioned anti-4-1BB scFv is oriented from the N-terminus to the C-terminus. The heavy chain variable region of the aforementioned anti-4-1BB antibody, Selectively, peptide linkers (second peptide linkers), and The variable region of the light chain of an anti-4-1BB antibody.

[0098] Furthermore, selectively, the bispecific antibody may include the following: (i) First polypeptide from N-terminus to C-terminus: anti-4-1BB scFv, Selectively, peptide linkers (first peptide linkers), and The heavy chain of the anti-B7-H4 antibody; and (ii) Second polypeptide containing the light chain of anti-B7-H4 antibody, The aforementioned anti-4-1BB scFv is oriented from the N-terminus to the C-terminus. The light chain variable region of the anti-4-1BB antibody, Selectively, peptide linkers (second peptide linkers), and The heavy chain variable region of the aforementioned anti-4-1BB antibody.

[0099] Furthermore, selectively, bispecific antibodies may include the following: (i) First polypeptide from N-terminus to C-terminus: Section 4-1BB scFv, Selectively, peptide linkers (first peptide linkers), and The heavy chain of the anti-B7-H4 antibody; and (ii) Second polypeptide containing the light chain of anti-B7-H4 antibody, The aforementioned anti-4-1BB scFv is oriented from the N-terminus to the C-terminus. The heavy chain variable region of the anti-4-1BB antibody, Selectively, peptide linkers (second peptide linkers), and The variable region of the light chain of an anti-4-1BB antibody.

[0100] In another embodiment, both the B7-H4 targeted moisture and the 4-1BB targeted moisture contained in the bispecific antibody may be full-length antibodies or antigen-binding fragments containing heavy chain CDRs, light chain CDRs, or combinations thereof, which can be linked to each other directly or via peptide linkers.

[0101] Given that each antibody can bind to 4-1BB (e.g., human 4-1BB) and B7-H4 (e.g., human B7-H4), a CDR sequence, or both VH (heavy chain variable region) and VL, in the present invention, the disclosed (light chain variable region) sequences may be “mixed and matched” to generate other anti-B7-H4 / anti-4-1BB binding bispecific molecules.

[0102] In other examples, both the B7-H4 targeted moisture and the 4-1BB targeted moisture may be full-length antibodies or antigen-binding fragments containing heavy chain CDRs, light chain CDRs, or combinations thereof.

[0103] In another embodiment, the bispecific antibody may be in a heterodimer form, comprising a first arm containing a pair of first heavy chains and first light chains that target one of B7-H4 and 4-1BB, and a second arm containing a pair of second heavy chains and second light chains that target the remaining one.

[0104] In one example, the full-length antibody may be in full-length immunoglobulin form (e.g., IgG, IgM, IgA, IgE, or IgD, e.g., human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv, single-chain antibody, sdFv, etc., as described above. For example, the full-length antibody may be in full-length human IgG form (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment may be scFv.

[0105] Peptide linker

[0106] The use of peptide linkers in bispecific antibodies can produce high antibody purity. Specifically, for high antibody purity, bispecific antibodies can contain peptide linkers between the heavy chain and scFv in the first polypeptide (first peptide linker) and / or between the heavy chain and light chain variable region in the scFv (second peptide linker).

[0107] In this invention, the term "peptide linker" may contain 1 to 100, particularly 2 to 50, arbitrary amino acids, and may contain any type of amino acid without any limitations. The peptide linker may contain, for example, Gly, Asn, and / or Ser residues, and may also contain neutral amino acids, such as Thr and / or Ala. Suitable amino acid sequences for the peptide linker may be those known in the relevant art.

[0108] Furthermore, the length of the peptide linker can be determined in various ways, within limits that do not affect the function of the polypeptide and / or scFv. For example, the peptide linker may be formed by comprising a total of approximately 1 to 100, approximately 2 to 50, or approximately 5 to 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) one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala.

[0109] In one concrete example, the peptide linker is (G m S1) n (m, 1, and n may independently be represented by constants 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, (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9 amino acids.

[0110] Variable antibodies

[0111] For example, the antibody of the present invention may include a flexible linker sequence or may be modified to add a functional moisture (e.g., PEG, drug, toxin, or label).

[0112] The antibodies or variants of the present invention may include derivatives modified by, for example, covalent attachment of any type of molecule to the antibody, where the covalent attachment does not prevent the antibody from binding to an antigen (e.g., an epitope). For example, the antibody may be modified by at least one selected from the group consisting of glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein hydrolysis cleavage, linkage to cell ligands or other proteins, etc.

[0113] Many chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tunicamycin. Additionally, the antibody may contain one or more non-classical amino acids.

[0114] For certain applications, including the in vivo use of antibodies in humans and in vitro detection and analysis, it is preferable to use chimeric, humanized, or human antibodies. Methods for producing chimeric or humanized antibodies are known in the art.

[0115] In another embodiment, the DNA encoding the desired antibody may be readily isolated and sequenced using standard procedures. Isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed in an expression vector and then plasma-infected by prokaryotic or eukaryotic host cells such as Bacillus malva cells, Simian COS cells, Chinese hamster ovary (CHO) cells, or other myeloma cells that do not produce immunoglobulins.

[0116] The present invention provides isolated polynucleotides that encode anti-B7-H4 / anti-4-1BB bispecific antibodies, anti-B7-H4 antibodies, anti-4-1BB antibodies, or antigen-binding fragments thereof.

[0117] In one embodiment, an expression vector containing isolated polynucleotides can be provided.

[0118] Furthermore, in one embodiment, hybridoma cells containing isolated polynucleotides that encode the antibody or antigen-binding fragment of the present invention or an expression vector containing the same can be provided.

[0119] Furthermore, non-human antibodies can be humanized by inserting one or more CDRs of an antibody into a framework region, such as a human framework region, using conventional recombinant DNA technology. The framework region may be a spontaneously occurring or consensus framework region, and preferably a human framework region (see, for example, Chothia et al., J.Mol.Biol.278:457~479 (1998) for a list of human framework regions). For example, the polynucleotide produced by the combination of a framework region and a CDR encodes an antibody, e.g., LIGHT, that specifically binds to at least one epitope of a desired polypeptide. Preferably, it may consist of one or more amino acid substitution framework regions, preferably the amino acid substitution improves the antibody's binding to the antigen (or epitope). Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in disulfide bonds in the chain, in order to produce antibody molecules that lack one or more disulfide bonds in the chain. Other modifications to polynucleotides are included within the scope of this disclosure and the art.

[0120] Additionally, mutations can be introduced into the nucleoside sequence that encodes the antibody of the present invention using standard techniques known to those skilled in the art.

[0121] The "heavy component" of the anti-B7-H4 / anti-4-1BB bispecific antibody of the present invention The "Component)" may include, if the bispecific antibody includes a full-length anti-B7-H4 antibody and an anti-4-1BB scFv, (1) the heavy chain of the anti-B7-H4 antibody and (2) the heavy chain variable region and light chain variable region of the anti-4-1BB antibody; or if the bispecific antibody includes a full-length anti-4-1BB antibody and an anti-B7-H4 scFv, (1) the heavy chain of the anti-4-1BB antibody and (2) the heavy chain variable region and light chain variable region of the anti-B7-H4 antibody.

[0122] The "light component" of the anti-B7-H4 / anti-4-1BB bispecific antibody of the present invention. The "Component)" may include: the light chain of the anti-B7-H4 antibody if the bispecific antibody includes a full-length anti-B7-H4 antibody and an anti-4-1BB scFv; or the light chain of the anti-4-1BB antibody if the bispecific antibody includes a full-length anti-4-1BB antibody and an anti-B7-H4 scFv.

[0123] Therapeutic uses of bispecific antibodies

[0124] The bispecific antibody of the present invention can simultaneously bind to B7-H4 protein and 4-1BB protein on the cell surface, and therefore can exhibit improved effects in immunotherapy and / or cancer therapy, for example, by activating the immune response in the tumor microenvironment.

[0125] Given the bispecific antibody of the present invention's ability to bind to the B7-H4 protein and stimulate antigen-specific T cell responses, the antibody of the present invention can be used to provide compositions or in vitro and in vivo methods for stimulating, enhancing, or modulating antigen-specific T cell responses.

[0126] One embodiment provides a pharmaceutical composition comprising the bispecific antibody or anti-B7-H4 or anti-4-1BB antibody. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to stimulate an immune response (e.g., antigen-specific T cell response) and / or to treat and / or prevent diseases associated with B7-H4, 4-1BB, or both.

[0127] Another example of its application is the use of bispecific antibodies or anti-B7-H4 antibodies in the prevention or treatment of diseases associated with B7-H4.

[0128] Another embodiment provides a method for treating and / or preventing a disease, such as cancer, related to B7-H4, 4-1BB, or both, by stimulating an immune response (e.g., an antigen-specific T cell response) in a subject requiring such treatment and / or prevention, comprising administering a pharmaceutically effective amount of a bispecific antibody or pharmaceutical composition to the subject. The subject may be a subject that needs to be treated and / or prevent a disease, such as cancer, related to B7-H4, 4-1BB, or both, by stimulating an immune response (e.g., an antigen-specific T cell response). The method may further include a step of identifying a subject that needs to be treated and / or prevent a disease related to B7-H4, 4-1BB, or both, prior to the administration step.

[0129] Another embodiment provides the use of the bispecific antibody or the therapeutic composition in treating and / or preventing diseases such as cancer, for example, related to stimulating an immune response (e.g., antigen-specific T cell response) and / or B7-H4, 4-1BB or both thereof. Another embodiment provides the use of the bispecific antibody in manufacturing agents for treating and / or preventing diseases such as cancer, for example, related to stimulating an immune response (e.g., antigen-specific T cell response) and / or B7-H4, 4-1BB or both thereof.

[0130] In one embodiment, the subject may be selected from mammals including, but not limited to, humans, monkeys, rhes, mice, dogs, cats, guinea pigs, rabbits, rhes, horses, cattle, cows, and others, or cells or organisms derived therefrom. For example, the subject may be one that needs to be treated and / or prevented from a disease such as cancer, which is related to immune response stimulation (e.g., antigen-specific T cell response) and / or B7-H4, 4-1BB or both. For example, the subject may be a mammal suffering from cancer (e.g., a human).

[0131] Another concrete example involves cells (separated or isolated) from mammals suffering from a disease selected from, for example, cancer, infectious disease, autoimmune reaction, or neurological disorder (e.g., cancer cells or cells isolated from an infected site in a mammal, or T cells, such as tumor-infiltrating T lymphocytes, CD4). + T cells, CD8 + This may be a T cell, or a combination thereof.

[0132] In one embodiment, the disease may be related to the expression or high expression (overexpression) of B7-H4. For example, the disease may be a cancer related to the expression or high expression (overexpression) of B7-H4. For example, "cancer related to high expression of B7-H4" can refer to a cancer associated with cancer cells that express more B7-H4 than non-B7-H4 cancer cells (e.g., cancer cell lines SK-BR3, CAMA-1, etc.).

[0133] In the pharmaceutical compositions, methods, and / or uses provided in the present invention, diseases related to B7-H4, 4-1BB, or both thereof may be diseases related to the activation (e.g., abnormal activation or hyperactivation) and / or overproduction (overexpression) of B7-H4, 4-1BB, or both thereof. For example, the disease may be cancer or infection.

[0134] The cancer may be a solid tumor or a hematological cancer. Specifically, the cancer may be one or more cancers selected from the group consisting of breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell carcinoma of the lung, small cell lung cancer, noncellular lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, melanoma of the skin and eyeballs, rectal cancer, perianal cancer, esophageal cancer, small intestinal tumor, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, late-onset or acute leukemia, lymphocytic lymphoma, liver cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, blepharocarcinoma, endometrial cancer or uterine cancer, salivary gland tumor, kidney cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, etc., but is not limited thereto. The cancer may be primary or metastatic.

[0135] In this invention, the term "prevention and / or treatment of cancer" can mean the death of cancer cells, the suppression of cancer cell proliferation, the alleviation of cancer-related symptoms, the suppression of cancer metastasis, and so on.

[0136] In the present invention, the term “enhancement of immune response” can mean any enhancement of an immune response related to 4-1BB, such as 4-1BB signaling activation or 4-1BB-induced signaling activation (e.g., 4-1BB-induced NF-κB signaling activation, increased cytokine release, target cell death by immune cells such as T cells, etc., but not limited to these). In one embodiment, the enhancement of immune response by a bispecific antibody provided by this disclosure can occur in the presence of B7-H4 (under conditions of B7-H4 expression).

[0137] Specific doses and therapeutic regimens for particular patients vary depending on a variety of factors, including the specific antibody, its variants or derivatives, the patient's age, weight, general health, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The amount used can be determined by pharmacological and pharmacokinetic principles well known in this field.

[0138] The administration of a bispecific antibody or an anti-B7-H4 or anti-4-1BB antibody may be carried out via one or more of the following methods: intraperitoneal vein, subcutaneous, intradermal, intramuscular, intranasal, epidural, and oral administration, but is not limited thereto.

[0139] Bispecific antibodies or anti-B7-H4 or anti-4-1BB antibodies or compositions can be administered by any convenient route, e.g., infusion or primary injection, along with other biologically active agents, in the epithelial or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Accordingly, the pharmaceutical compositions containing the antigen-binding polypeptide of the present invention can be administered orally, parenterally, intracellularly, intravaginally, intraperitoneally, rectally, topically (by powder, ointment, drip or oral patch), sphincterally, or orally or sphincterally, or by oral or nasal spray. In the present invention, the term “parenteral” means a method of administration including intravenous, intramuscular, intraperitoneal, intrasternal, intratumoral, subcutaneous and intra-articular injections and infusions.

[0140] To treat, suppress, improve and / or treat inflammatory, immune, or malignant diseases, disorders, or conditions. The pharmaceutically effective dose of a bispecific antibody or anti-B7-H4 or anti-4-1BB antibody for prevention can be determined by standard clinical techniques.

[0141] The pharmaceutical compositions of the present invention may contain an effective amount of a bispecific antibody or an anti-B7-H4 or anti-4-1BB antibody and an acceptable carrier. In some embodiments, the composition may further contain a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0142] Diagnostic uses of antibodies

[0143] Overexpression and / or hyperactivation of B7-H4 and / or 4-1BB have been observed in biological samples (e.g., cells, tissues, blood, serum, etc.) from patients suffering from certain cancers and / or infections (e.g., tissues, blood, or serum from tumor cells or infected patients), or patients with B7-H4 and / or 4-1BB overexpressing cells may respond to treatment with bispecific antibodies or anti-B7-H4 or anti-4-1BB antibodies. Therefore, the bispecific antibodies or anti-B7-H4 or anti-4-1BB antibodies of the present invention can be used for diagnostic and prognostic purposes.

[0144] One example is the provision of a pharmaceutical composition for diagnosing diseases related to B7-H4, 4-1BB, or both, which comprises a bispecific antibody or an anti-B7-H4 or anti-4-1BB antibody.

[0145] Other embodiments provide applications for bispecific antibodies or anti-B7-H4 or anti-4-1BB antibodies for diagnosing diseases associated with B7-H4, 4-1BB, or both. In diagnostic and / or detection methods, the step of detecting an antigen-antibody reaction or measuring the level of an antigen-antibody reaction may be carried out by any common method known in the relevant art.

[0146] Another example provides a method for providing diagnostic information regarding diseases related to B7-H4.

[0147] Polynucleotides for encrypting antibodies and methods for producing antibodies

[0148] One embodiment of the present invention provides a polynucleotide for encoding a bispecific antibody or an anti-B7-H4 or anti-4-1BB antibody. Specifically, one embodiment provides a polynucleotide for encoding the heavy chain of a bispecific antibody in IgG-scFv form.

[0149] Another embodiment provides a polynucleotide that encodes the light chain of a bispecific antibody in the IgG-scFv form. The IgG-scFv form can refer to a type of bispecific antibody comprising a full-length IgG antibody that targets (binds to) one of the B7-H4 and 4-1BB proteins and an scFv fragment that targets (binds to) the other, where the scFv can be ligated directly (without a peptide linker) or via a peptide linker to the C-terminus and / or N-terminus of the full-length IgG antibody.

[0150] In one embodiment, if a bispecific antibody in IgG-scFv form contains a full-length IgG antibody against B7-H4 and an scFv fragment against 4-1BB, the polynucleotide encoding the heavy chain of the bispecific antibody can encode the heavy chain of the full-length IgG antibody against B7-H4 and the scFv fragment against 4-1BB linked directly to the C-terminus and / or N-terminus of the full-length IgG antibody or via a peptide linker, and the polynucleotide encoding the light chain of the bispecific antibody can encode the light chain of the full-length IgG antibody against B7-H4.

[0151] In another embodiment, if a bispecific antibody in IgG-scFv form contains a full-length IgG antibody against 4-1BB and an scFv fragment against B7-H4, the polynucleotide encoding the heavy chain of the bispecific antibody can encode the heavy chain of the full-length IgG antibody against 4-1BB and the scFv fragment against B7-H4, which is directly or via a peptide linker linked to the C-terminus and / or N-terminus of the full-length IgG antibody, and the polynucleotide encoding the light chain of the bispecific antibody can encode the light chain of the full-length IgG antibody against 4-1BB.

[0152] Another example provides a recombinant vector containing a polynucleotide that encodes the heavy chain of a bispecific antibody, a polynucleotide that encodes the light chain of a bispecific antibody, or both. Yet another example provides recombinant cells that have been plasma-infected with the recombinant vector.

[0153] In one embodiment, the expression vector comprises isolated polynucleotides that encode the antibody or antigen-binding fragment of the present invention.

[0154] Another embodiment provides a method for producing a bispecific antibody, which includes expressing polynucleotides that encode the heavy chain of the bispecific antibody and polynucleotides that encode the light chain of the bispecific antibody in cells.

[0155] The step of expressing polynucleotides may be carried out by culturing cells containing polynucleotides (e.g., recombinant vectors) under conditions that allow for polynucleotide expression. The method may further include a step of separating and / or purifying bispecific antibodies from the cell culture after the expression or culture step. [Effects of the Invention]

[0156] This invention relates to a bispecific antibody comprising a B7-H4-specific antibody and a 4-1BB-specific antibody, respectively, and its applications. The anti-B7-H4 / 4-1BB bispecific antibody can have a high affinity for B7-H4 and / or 4-1BB, and can enhance the immune response and / or treat mammalian tumors (cancer) while reducing hepatotoxicity.

[0157] Furthermore, the anti-B7-H4 monospecific antibody of the present invention exhibits the function of suppressing the checkpoint activity of B7-H4 by specifically binding to and detecting B7-H4 expressed in cancer cells with high affinity. [Brief explanation of the drawing]

[0158] [Figure 1] Figure 1 shows the antigen-binding ability of mouse anti-B7-H4 antibody as determined by FACS analysis. [Figure 2] Figure 2 shows the antigen-binding capacity of the humanized antibody 16E3H3 M1 as measured by ELISA. [Figure 3] Figure 3 shows the results of FACS analysis of the cancer cell binding characteristics of the anti-B7-H4 antibody. [Figure 4] Figure 4 shows the results of measuring the T-cell checkpoint ligand activity of the anti-B7-H4 antibody by measuring IFN-γ secretion from T cells. [Figure 5] Figure 5 shows the results of measuring the T-cell checkpoint ligand activity of the anti-B7-H4 antibody in a dose-dependent manner by measuring IFN-γ secretion from T cells. [Figure 6]Figure 6 shows the antigen-binding ability of anti-B7-H4 / anti-4-1BB bispecific antibodies against human B7-H4 and 4-1BB proteins, as measured by DACE. [Figure 7] Figure 7 shows the antigen-binding ability of anti-B7-H4 / anti-4-1BB bispecific antibodies against monkey B7-H4 and 4-1BB proteins as measured by DACE. [Figure 8] Figure 8 shows the cancer cell binding characteristics of the anti-B7-H4 / anti-4-1BB bispecific antibody as determined by FACS analysis. [Figure 9] Figure 9 shows the results of measuring the binding ability of anti-B7-H4 / anti-4-1BB bispecific antibodies to B7-family proteins by ELISA. [Figure 10] Figure 10 shows the results of measuring the T cell checkpoint ligand activity of the anti-B7-H4 / anti-4-1BB bispecific antibody by measuring IFN-γ secretion from T cells. [Figure 11] Figure 11 shows the results of measuring the T cell checkpoint ligand activity of the anti-B7-H4 / anti-4-1BB bispecific antibody in a dose-dependent manner by measuring IFN-γ secretion from T cells. [Figure 12] Figure 12 shows the results of a PBMC-based targeted cell lysis test using anti-B7-H4 / anti-4-1BB bispecific antibodies. [Figure 13a] Figure 13a shows the results of PBMC-based target cell lysis studies using anti-B7-H4 / anti-4-1BB bispecific antibodies with various PBMC lots. [Figure 13b] Figure 13b shows the results of PBMC-based target cell lysis studies using anti-B7-H4 / anti-4-1BB bispecific antibodies with various PBMC lots. [Figure 14] Figure 14 shows the in vitro 4-1BB activity test results for the anti-B7-H4 / anti-4-1BB bispecific antibody. [Figure 15] Figure 15 shows the results of a dose-dependent in vivo efficacy study (antitumor activity) of the anti-B7-H4 / anti-4-1BB bispecific antibody. [Figure 16] Figure 16 shows the results of a dose-dependent in vivo efficacy study (antitumor activity) of the anti-B7-H4 / anti-4-1BB bispecific antibody. [Figure 17] Figure 17 shows the results of a dose-dependent re-challenge study (antitumor activity) of the anti-B7-H4 / anti-4-1BB bispecific antibody. [Examples]

[0159] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited to the following examples.

[0160] Production example 1. Anti-B7-H4 antibody generation

[0161] 1-1. Generation of mouse monoclonal antibodies by immunization

[0162] Monoclonal antibodies against human B7-H4 were generated using the following procedure: SJL and Balb / C mice (Charles River Laboratories, Hollister, CA) were hyperimmunized with one of 293 cells overexpressing recombinant human B7-H4.

[0163] Specifically, mice were injected with 293 cells overexpressing human B7-H4 in PBS (5 million cells / dose via intraperitoneal infusion), followed by injection of protein booster along with recombinant human B7-H4ECD (4 Dg / dose via intraperitoneal infusion). Serum titers were assessed after 6–9 injections by standard enzyme-linked immunosorbant assay (ELISA) and FACS. Spleen B cells isolated from serum B7-H4-positive mice were fused with mouse myeloma cells (X63.Ag8.653; American Type Culture Collection, Monesos, VA, USA) by electrofusion (Hybrimune; Harvard Life Sciences, Holliston, MA, USA). After 10–14 days, the hybridoma supernatant was analyzed for antibody secretion by ELISA. Screening was performed. Subsequently, all positive clones were expanded and further screened for binding to huB7-H4 by ELISA and FACS.

[0164] Three hybridoma clones were identified: 16E3H3, 23B6C2, and 73B4F10 (identified from B7-H4 immunized SJL mice). As shown in Figure 1, all clones positively bound to B7-H4 expressing cell lines (CHOK-1 B7-H4, SK-BR-3) and recombinant human B7-H4 by fluorescence-activated cell classification (FAC). The variable regions and CDRs of the identified mouse clones are as follows:

[0165] [Table 9]

[0166] [Table 10]

[0167] [Table 11]

[0168] 1-2. Cloning and Chimerization of Mouse Monoclonal Antibodies

[0169] The mouse monoclonal antibodies 16E3H3, 23B6C2, and 73B4F10 were cloned and chimeric as described below.

[0170] Total RNA was extracted from hybridoma cells producing Mullin 16E3H3, 23B6C2, and 73B4F10 using standard methods. Variable light chain (VL) and variable heavy chain (VH) domains were amplified by RT-PCR with degenerate primers for the heavy and light chains, respectively. Forward primers were specific to the N-terminal amino acid sequences of the VL and VH regions, while LC and HC reverse primers were designed to anneal to the highly conserved, species-wide invariant light (CL) and invariant heavy domain 1 (CH1) regions.

[0171] The polynucleotide sequences of the inserts were determined using standard sequencing methods. The mouse heavy chain variable region was cloned into the human IgG1 heavy chain invariant region, and the light chain variable region was cloned into the human kappa light chain invariant region to create chimeric antibodies.

[0172] 1-3. Humanization of mouse monoclonal antibodies

[0173] The structure of the parent antibody was modeled using a computer-assisted homology modeling program. Humanized antibodies were designed using CDR transplantation + reverse mutation. Briefly, the CDR of the parent antibody was transplanted into a human receptor to obtain a humanized light chain. Three (3) chimeric claws Of these, 16E3H3 was selected as the dominant clone. The sequences of the humanized heavy and light chains of the 16E3H3 clone are shown in Tables 12 and 13.

[0174] The light chain was further modified to remove post-translational modification (PTM). The modified light chain sequence is shown in Table 14 (16E3H3 M1 clone). The invariant region of the antibody contained in the antibody can continue to be modified by introducing one or more mutations or alterations (e.g., ADCC-decrease mutation (N297A mutation; cancer cells, vol. 19, issue 1, pp. 101-113, etc.)) into human IgG1.

[0175] [Table 12]

[0176] [Table 13]

[0177] [Table 14]

[0178] Manufacturing Example 2. Production of anti-4-1BB antibody

[0179] 2-1. Production of fully human anti-4-1BB monoclonal antibody

[0180] A complete human anti-4-1BB monoclonal antibody was manufactured as disclosed in WO2020 / 111913. Specifically, a total of four pannings were performed using 4-1BB (NCBI registry number NP_001552.2) coated immunotubes for panning a phage library (obtained from KBio Health) against the target molecule. Bacterial colonies from the results of three pannings were grown in 96 deep-well plates with SB-carbenicillin until turbid, and at this point 10 11 Pfu-containing VCSM13 helper phage was added to each well. After infecting the cells with gentle shaking at 37°C for 1 hour (80 rpm), 70 μg / mL of kanamycin was added, and the cells were cultured overnight at 30°C with shaking at 200 rpm. The following day, the plate was centrifuged, and the supernatant containing the phage was added to an ELISA plate coated with 4-1BB antigen blocked with 3% BSA in PBST. After incubation at room temperature for 1 hour, the plate was washed three times with PBST, and anti-M13 antibody was added. The plate was cultured for 1 hour, washed three times with PBST, and the binding activity was measured using tetramethylbenzidine (TMB).

[0181] The 4-1BB-specific binder was amplified for plasmid DNA sequencing. The Ig light chain V gene (VL) and VH sequences were analyzed to confirm unique sequences and determine sequence diversity.

[0182] 2-2. Production of anti-4-1BB scFv antibody

[0183] The anti-4-1BB scFv antibody having the structure (N')-VL-linker-VH-(C') was prepared using the variable region of the complete human monoclonal antibody against 4-1BB obtained in Production Example 2-1, where the 44th amino acid residue "G" in the heavy chain variable region was replaced with "C", and the 103rd amino acid residue "G" in the light chain variable region was replaced with "C". Such amino acid substitution from "G" to "C" in scFv can contribute to increased safety of a bispecific antibody containing scFv as one target-specific moiety. The amino acid sequences of the prepared anti-4-1BB scFv are shown in Tables 15 to 21 below, and those skilled in the art can apply changes or modifications to the amino acid sequence to satisfy specific purposes, including the application of various types of peptide linkers such as (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9.

[0184] [Table 15]

[0185] [Table 16]

[0186] [Table 17]

[0187] [Table 18]

[0188] [Table 19]

[0189] [Table 20]

[0190] [Table 21]

[0191] [Table 22]

[0192] 2-3. Production of anti-4-1BB scFv antibody in the form of a bispecific antibody

[0193] As a typical example of a bispecific antibody, we prepared an anti-B7-H4x4-1BB bispecific antibody composed of a heavy and a light composition, as shown below.

[0194] (1) Heavy component (N'→C') 1) Heavy chain of anti-B7-H4 antibody; 2) Linker: (GS)9; and 3) Anti-4-1BB scFv prepared in manufacturing example 2-2 (2) Lighter component (N'→C') Light chain of anti-B7-H4 antibody

[0195] Manufacturing Example 3. Production of anti-B7-H4 / anti-4-1BB bispecific antibody To produce an anti-B7-H4 / anti-4-1BB bispecific antibody in an IgG-scFv fusion form, the anti-B7-H4 IgG and anti-4-1BB scFv clones produced in Production Example 1 and Production Example 2 were respectively exemplarily selected, where the scFv antibody fragment of one antigen is fused to the C-terminus of the IgG of the other antigen. When B7-H4 is located in the whole IgG portion, IgG1 (N297A mutation) having a mutant backbone with reduced ADCC was used, and when 4-1BB is located in the whole IgG portion, IgG4 was used.

[0196] In particular, anti-B7-H4 / anti-4-1BB bispecific antibody candidates were produced in the full-length IgG (anti-B7-H4 antibody)-scFv (anti-4-1BB antibody) format. The constant region of the anti-B7-H4 antibody contained in the bispecific antibody was modified by introducing the NA mutation (N297A). The amino acid sequences of the bispecific antibodies (16E3H3 x1A10 M12 and 16E3H3 M1 x1A10 M12) produced by this production example are shown in Table 23 and Table 24 respectively.

[0197] Hereinafter, the 16E3H3 M1 monospecific antibody is also referred to as M40413, and 16E3H3 M1 x1A10 M12 bispecific antibody is also referred to as B10317.

[0198]

Table 23

[0199]

Table 24

[0200] Cloning of the bispecific antibody was performed as follows. DNA segment 1 having a nucleoside sequence encoding the heavy chain of the IgG antibody of the anti-B7-H4 / anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1), and DNA segment 2 having a nucleoside sequence encoding the light chain of the IgG antibody of the anti-B7-H4 / anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 2). Thereafter, to produce a vector for the expression of the bispecific antibody, DNA segment 4 encoding a linker peptide having a length of 16 amino acids composed of (GGGGS)4 (SEQ ID NO: 68) or DNA segment 5 encoding a linker peptide having a length of 18 amino acids composed of (GS)9 (SEQ ID NO: 69) was used to fuse DNA segment 3 encoding scFv to a part of DNA segment 1 corresponding to the c-terminus of the Fc region of IgG inserted into plasmid 1. Also, as described in Production Example 2 to stabilize scFv, additional modifications were applied to generate disulfide bridges by fusing VL103-VH44 (VL103: VL having a G→C mutation at the 103rd position; VH44: VH having a G→C mutation at the 44th position) to the C-terminus of the light chain and the C-terminus of the heavy chain, respectively.

[0201] Experimental Example 1. Antigen-binding ability of the anti-B7-H4 antibody

[0202] 1-1. Confirmation of the binding of the mouse anti-B7-H4 antibody by using FACS analysis

[0203] To evaluate antigen-binding properties, the binding of mouse antibody candidate substances produced by Production Example 1-1 to B7-H4 expressing cells (SK-BR3, CAMA-1, and MDA-MB-468) or B7-H4 negative expressing cells (PANC-1) was analyzed. Briefly, each cell was treated with the presented antibody at 100 nM at 4°C for 1 hour. After washing with FACS buffer, the cells were incubated with FITC anti-mouse IgGFc antibody at 4°C for 1 hour, and then FACS analysis was performed. As shown in Figure 1, the anti-B7-4H mouse antibody bound to the B7-H4 expressing SK-BR3, CAMA-1, and MDA-MB-468 cell lines, but not to B7-H4 negative PANC-1. As a result, of the three (3) mouse antibody candidate substances, the 16E3H3 clone showed the highest affinity to B7-H4 expressing cancer cells. Based on the ranking of the results, 16E3H3 was selected for humanization.

[0204] 1-2. Antigen-binding ability of anti-B7-H4 monospecific antibodies against human B7-H4 protein

[0205] To evaluate the antigen-binding activity of humanized antibodies, ELISA was performed against 16E3H3 M1. Briefly, ELISA plates were coated overnight at 4°C with 1 ug / ml, 100 ul / well of Fc-fused human B7-H4 protein in PBS, and then blocked at 37°C with 200 ul / well of 1% BSA. A 4-fold dilution of a single clone antibody, starting at 100 nM, was added to each well and incubated at 37°C for 1 hour. The plates were washed with PBST (0.05% Tween 20 in PBS) and incubated with anti-human Fab conjugated with HRP (Horse Radish Peroxidase) at 37°C for 1 hour. The plates were then washed with PBST (0.05% Tween 20 in PBS) developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm to 650 nm. As shown in Figure 2, 16E3H3 M1 bound to B7-H4 in a dose-dependent manner. 50 The value was 0.079 nM.

[0206] Experimental Example 2. Cell-binding capacity of anti-B7-H4 monospecific antibody

[0207] To evaluate tumor antigen binding properties, 16E3H3 M1 clones were analyzed by FACS for their binding to B7-H4-expressing mammalian cells. Briefly, B7-H4 positive cells (MX-1, CAMA-1, SK-BR3, and OVCAR-3) or B7-H4 negative cells (PANC-1) were incubated with M40413 antibody. After washing with FACS buffer (PBS with 1% BSA), FITC-anti-human IgG antibody was added to each well and incubated at 4°C for 45 minutes. MFI of FITC was assessed using FACS Calibur. As shown in Figure 3, 16E3H3 M1 clones bound to B7-H4 positive cancer cell lines in a dose-dependent manner.

[0208] Experimental Example 3. T cell checkpoint ligand activity of anti-B7-H4 monospecific antibody measured by IFN-γ production.

[0209] B7-H4 suppression of T-cell activation was performed using plate-fixed recombinant B7-H4 protein (rB7-H4) and healthy donor PBMCs. Normal donor PBMC cells were subjected to I in the presence of control protein (hIgG1) or soluble rB7-H4. γ was stimulated with anti-CD3 antibody along with BMUR (Urelumab, BMS's anti--4-1BB antibody), M40413 (16E3H3 M1), or a reference B7-H4 antibody (FPA150, fiveprime's reference antibody) (Figure 4).

[0210] rB7-H4 clearly demonstrated T cell checkpoint ligand activity. This is evident from the graph in Figure 4, which shows that IFN-γ secretion from T cells was reduced in rB7-H4-coated plates compared to IgG-coated plates. On the other hand, M40413 (slash pattern in the graph) and FPA150 (vertical pattern in the graph) blocked the T cell checkpoint ligand activity of rB7-H4, which is supported by increased IFN-γ secretion levels compared to IgG (gray in the graph) or BMUR (white in the graph) treated conditions.

[0211] In other words, under B7-H4-mediated immunosuppressive conditions, other immunomodulatory mechanisms, such as anti-4-1BB antibodies, do not function. In contrast, these B7-H4-mediated immunosuppressions can be overcome by anti-B7-H4 antibodies. Furthermore, B7-H4-mediated T-cell suppression was more efficiently rescued by the B7-H4 antibodies of the present invention than by antibodies that are not specific to B7-H4 (T-cell activity recovery rate: M40413 > FPA150 > BMUR).

[0212] Experimental Example 4. Dose-dependent measurement of T cell checkpoint ligand activity of anti-H7-H4 antibody.

[0213] To characterize the T-cell checkpoint blocking activity of B7-H4 antibodies against stimulated human peripheral blood mononuclear cells (PBMCs), the concentration of IFN-gamma was measured in the supernatant secreted from T cells. B7-H4 suppression of T-cell activation was performed using plate-fixed recombinant B7-H4 protein and healthy donor PMBCs. Various concentrations of human IgG, BMUR (anti-4-1BB antibody from BMS), and B7-H4 antibody-M40413 or FPA150- were tested with PBMCs under constant temperature treatment in the presence of anti-human CD3. After 72 hours of incubation in a humidified chamber at 5% CO2 and 37°C, the IFN-gamma concentration in the supernatant was measured using the human IFN-gamma Quantikilne kit (R&D System, SIF50).

[0214] As shown in Figure 5, M40413 (circle in the graph) and FPA150 (inverted triangle in the graph) block the T-cell checkpoint ligand activity of rB7-H4, which is supported by increased IFN-γ secretion levels compared to IgG (empty square in the graph) or BMUR (diamond in the graph) treatment conditions. In other words, under B7-H4-mediated immunosuppressive conditions, other immunomodulatory measures, such as anti-4-1BB antibodies, do not function. In contrast, these B7-H4-mediated immunosuppressions can be overcome by anti-B7-H4 antibodies. Along with this, B7-H4-mediated T-cell suppression was more efficiently rescued by the B7-H4 antibodies of the present invention than by antibodies that are not specific to B7-H4.

[0215] Experimental Example 5. Relative expression estimation of cell surface B7-H4 using Dako's antibody dose kit.

[0216] Cell surface B7-H4 expression levels were quantified in various cancer cell lines using the QIFIKIT quantitative kit (Dako) as recommended by the manufacturer. Briefly, cells were stained at saturation concentration with unlabeled anti-B7-H4 mouse monoclonal antibody (abcam) or purified mouse IgG1k isotype control (abcam). After washing, the kit-stained cells and correction beads were simultaneously labeled with the same FITC-conjugated goat anti-mouse IgG secondary antibody from the kit. The labeled cells and correction beads were analyzed by flow cytometry. Linear regression was performed using the MFI value of the correction beads. ABC (antibody-binding capacity) was extrapolated from this regression line, and sABC (specific ABC) was calculated as follows: The sABCs were determined by skipping the ABCs of the isotype control antibody from the ABCs of the anti-B7-H4 antibody. As shown in Table 25, the sABCs of 11 cancer cell lines were determined. CHOK1, PANC1, and MC38 were considered B7-H4 negative cell lines.

[0217] [Table 25]

[0218] Experimental Example 6. Antigen-binding ability of anti-B7-H4 / anti-4-1BB bispecific antibody against human B7-H4 and 4-1BB proteins

[0219] To evaluate the antigen-binding activity, DACE was performed on the bispecific antibody B10317 (16E3H3 M1 x1A10 M12). Briefly, microtiter plates were coated overnight at 4°C with 1 μg / ml of human 4-1BB-Fc protein in PBS at 100 μl / well, and then blocked at 37°C with 200 μl / well of 1% BSA in PBS. Serial dilutions of the bispecific antibody starting from 100 nM were added to each well and incubated at 37°C for 1 hour. After washing the plates with PBS / Tween, they were incubated at 37°C for 1 hour with 1% BSA in PBS containing 1 μg / ml of human B7-H4his protein. After washing the plates with PBST (0.05% Tween 20 in PBS), they were incubated at 37°C for 1 hour with HRP-conjugated anti-his antibody. After washing, the plates were developed with TMB substrate and analyzed with a spectrophotometer at 450 nm - 650 nm. As shown in Figure 6, B10317 bound simultaneously to both human B7-H4 and human 4-1BB in a dose-dependent manner. The EC 50 (nM) value was 1.2 nM.

[0220] Experimental Example 7. Antigen-binding ability of anti-B7-H4 / anti-4-1BB bispecific antibody against cynomolgus monkey B7-H4 and 4-1BB proteins

[0221] To evaluate the cross-reactivity of bispecific antibodies, DACE was performed on the bispecific antibodies. Briefly, micro-optimal plates were coated with Rhesus 4-1BB-Fc protein (100 ng / well) overnight at 4°C, and then blocked with 200 μl / well of PBSB (PBS with 1% BSA). A 3-fold dilute solution of B10317 from 100 nM was added to each well and incubated at 37°C for 1 hour. The plates were washed with PBST (PBS with 0.05% Tween 20) and then incubated with Rhesus B7-H4-his protein (100 ng / well) at 37°C for 1 hour. The plates were washed with PBST (PBS with 0.05% Tween 20) and then incubated with HRP (Horse Radish Peroxidase) conjugated with anti-his antibody at 37°C for 1 hour. After washing, the plates were incubated with HRP (Horse Radish Peroxidase) at 37°C for 1 hour. The samples were developed with TMB substrate and analyzed by spectrophotometer at 450 nm to 650 nm. As shown in Figure 7, B10317 simultaneously bound to Rhesus B7-H4 and 4-1BB in a dose-dependent manner. 50 The (nM) value was 2.58 nM.

[0222] Experimental Example 8. Cell Binding Ability of Anti-B7-H4 / Anti-4-1BB Bispecific Antibodies

[0223] To evaluate the tumor antigen binding properties, the binding of a bispecific antibody to B7-H4-expressing mammalian cells was analyzed by FACS. Briefly, B7-H4-positive cells (MX-1 and CAMA-1) were incubated with the B10317 antibody. After washing with FACS buffer (PBS with 1% BSA), the FITC-anti-human IgG antibody was added to each well, and the cells were incubated at 4°C for 45 minutes. The MFI of FITC was assessed using FACS Calibur. As shown in Figure 8, B10317 bound to B7-H4-positive cancer cell lines in a dose-dependent manner.

[0224] Experimental Example 9. Binding affinity (SPR) of anti-B7-H4 / anti-4-1BB bispecific antibodies to target proteins B7-H4 and 4-1BB.

[0225] In the SPR experiment, the anti-B7-H4 / anti-4-1BB bispecific antibody obtained in Production Example 3 was captured in flow cells 2, 3, and 4, with flow cell 1 maintained as a reference. The anti-B7-H4 / anti-4-1BB bispecific antibody was immobilized on the Protein A tip by amine coupling. Recombinant B7-H4 (human / monkey) or 4-1BB (human / monkey) protein was flowed across the tip at a concentration ranging from 100 nM to 6.25 nM for B7-H4 or 250 nM to 15.625 nM for 4-1BB at 30 μl / min for 60 seconds, followed by a 180-second release step. Regeneration was performed using 10 mM glycine-HCl (pH 1.5). The results are shown in Table 26. As shown in Table 26, B10317 exhibits high affinity for B7-H4 and 4-1BB, with similar affinity for their respective human and monkey targets (B7-H4 or 4-1BB).

[0226] [Table 26]

[0227] Experimental Example 10. Protein Binding Test for B7-Family Proteins

[0228] To confirm the specific binding of anti-B7-H4 / anti-4-1BB bispecific antibodies to B7-H4, an ELISA test was performed targeting B10317. Microscale plates were coated with each protein (100 ng / well) overnight at 4°C, and then blocked with 200 μl / well of PBSB (1% (w / v) BSA in PBS). A 3-fold dilution of B10317, starting at 100 nM, was added to each well and incubated at 37°C for 1 hour. The plates were washed with PBST (0.05% (v / v) Tween 20 in PBS) and then incubated with 100 μl of anti-FcHRP (dilution ratio, 1:30000) or anti-Fab HRP (dilution factor, 1:20000) at 37°C for 1 hour. The plates were incubated for a specified time. After washing the plates with PBST (0.05% (v / v) Tween 20 in PBS), the plates were colored with TMB and analyzed by spectrophotometer at 450-650 nm. As shown in Figure 9, it was confirmed that B10317 specifically binds to B7-H4 and does not bind to other B7-family proteins.

[0229] Experimental Example 11. T cell checkpoint ligand activity of anti-B7-H4 / anti-4-1BB bispecific antibody measured by IFN-γ production.

[0230] B7-H4 suppression of T-cell activation was performed using plate-fixed recombinant B7-H4 protein (rB7-H4) and healthy donor PBMCs. Normal donor PBMC cells were stimulated with anti-CD3 antibodies along with IgG, B10317 bispecific antibody, Keytruda (anti-PD-1 antibody from MSD), M40413 monospecific antibody, or reference B7-H4 antibody, in the presence of control protein (hIgG1) or soluble rB7-H4 (Figure 10).

[0231] rB7-H4 clearly demonstrated T-cell checkpoint ligand activity. As shown in Figure 10, IFN-γ secretion from T cells was reduced in rB7-H4 coated plates compared to IgG-coated plates. On the other hand, B10317 (gray in the graph), M40413 (slash pattern in the graph), and FPA150 (vertical pattern in the graph) blocked the T-cell checkpoint ligand activity of rB7-H4, which is supported by increased IFN-γ secretion levels compared to IgG (black in the graph) or Keytruda (white in the graph) treated conditions.

[0232] In other words, under B7-H4-mediated immunosuppressive conditions, other immunomodulatory mechanisms, such as anti-PD-1 antibodies, do not function. In contrast, such B7-H4-mediated immunosuppression can be overcome by anti-B7-H4 antibodies according to the present disclosure. Furthermore, B7-H4-mediated T-cell suppression was rescued more efficiently by the monospecific or bispecific B7-H4 antibodies of the present invention than by control antibodies (T-cell activity recovery rate: B10317 > M40413 ≈ FPA150).

[0233] Experimental Example 12. Dose-dependent T cell checkpoint ligand activity of anti-B7-H4 / anti-4-1BB bispecific antibodies in diverse PBMC lot co-culture systems.

[0234] Using the same method as in Experimental Example 4, IFN-gamma concentrations in the supernatant were measured in various PBMC lot co-culture systems to confirm the T cell checkpoint blocking activity of B10317. As shown in Figure 11, the levels of IFN-gamma in the supernatant differed among PBMC lots, but the IFN-gamma secretion levels increased in a dose-dependent manner.

[0235] Experimental Example 13. PBMC-based targeted cell lysis study using anti-B7-H4 / anti-4-1BB bispecific antibodies.

[0236] Human PBMCs were co-cultured with the SK-BR3 cell line (expressing human B7-H4) in the presence of anti-human CD3 antibody and test antibody. Briefly, PBMCs (lot number .HHU20190711) were cultured at 3.0 × 10⁶ per well. 4 Plated with SK-BR3, SK-BR3 has 1.0 x 10 per well. 4 Co-plating was performed (E:T ratio = 1:3). Bispecific antibody B10317 (starting at 40 nM and diluting 4-fold), reference antibody FPA150, urelumab (anti--4-1BBmAb from BMS), or a combination of anti--B7-H4 (M40413) and anti--4-1BB (1A10M12) monospecific antibodies (starting at 40 nM and diluting 4-fold) was added to each plate well. After 6 days of culture, the % of target cell lysis was measured using cell counting kit-8 (CCK-8, Dojindo_CK04-20). As shown in Figure 12, the anti-B7-H4 / anti-4-1BB antibody (B10317) more efficiently induced dose-dependent target cell lysis than Urelumab (anti-4-1BB antibody), FPA150, or the combination treatment group (anti-B7-H4 and anti-4-1BB monospecific antibodies). Using the same method as above, the target cell lysis ability of B10317 was tested with various PBMC lots, and the results are shown in Figures 13a (SK-BR3) and 13b (CAMA-1). As shown in Figures 13a and 13b, B10317 induced dose-dependent target cell lysis even when using various PBMC lots.

[0237] Experimental Example 14. In vitro 4-1BB activation test of bispecific antibodies under diverse B7-H4 expression conditions.

[0238] The in vitro 4-1BB activity of the anti-B7-H4 / 4-1BB bispecific antibody B10317 was analyzed using the Promega kit system. In a simplified manner, various cell lines (MC38-hB7-H4: 2.5 × 10⁶) were sampled in 100 μL of white 96-well analysis plates. 4 CAMA-1: 2.5×10 4 SK-BR3: 2.5 x 10 4 HCC1806-hB7-H4: 2.5×104 OVCAR3:2.5×10 4 HCC1954: 2.5 × 10 4 CHOK1:2.5×10 4 Panc-1: 2.5 × 10 4 and MC38: 2.5×10 4 ) was plated.

[0239] Cells were cultured overnight in a 37°C incubator with 5% CO2 humidification. After overnight culture, 100 μL of culture medium was removed and 25 μL of analytical medium (1% FBS containing RPMI1640) was added to the pre-plate target cells. 25 μL of B10317 (starting at 50 nM and diluting 3-fold) or BMUR (starting at 133 nM and diluting 6-fold) was added to the plate. The GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line was harvested and resuspended in analytical medium. 25 μL of the GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line was added to each well, with 2.5 × 10⁶ cells per well. 4 Cell plates were prepared. Cells were cultured for 6 hours in a 37°C incubator with 5% CO2 humidification. During incubation, the Bio-Glo™ reagent was reduced according to the manufacturer's guidelines. After 6 hours of incubation, 75 μL of Bio-Glo™ reagent per well was added to the analysis plate. After 5 minutes, luminescence was measured using a microplate reader. Four-parametric logistic curve analysis was performed using GraphPad software.

[0240] As shown in Figure 14, B10317 dose-dependently activated 4-1BB signaling only in the presence of B7-H4, and the T-cell stimulating activity of B10317 was related to the B7-H4 expression level. B10317 did not activate 4-1BB signaling in the absence of B7-H4 expression. On the other hand, T-cell stimulating activity increased in both B7-H4 positive and negative cell lines in the BMUR-treated group. From these results, it can be seen that B10317, unlike BMUR, particularly increases T-cell activity in tumor environments that express B7-H4.

[0241] Experimental Example 15. Evaluation of effective dose of B-CAG-hB7-H4 MC38 bispecific antibody containing humanized 4-1BB mice.

[0242] To evaluate the in vivo efficacy of the B10317 bispecific antibody, B-h4-1BB TG mice (Biocytogen) were subjected to tumor development by ingesting B-CAG-hB7-H4 MC38 tumor cells (1 × 10⁶) suspended in 0.1 mL of PBS. 6 The drug was subcutaneously injected into the entire right side. The tumor-bearing animals had a tumor size of 103 mm. 3 Upon arrival, participants were randomly enrolled in five research groups. Each group consisted of eight mice. The five groups were: G1 (hIgG1 isotype control group, 10 mg / kg), G2 (BMUR, 7.5 mg / kg, same molar ratio as B10317 at 10 mg / kg), and G3 (B103 The test doses were 17 mg / kg, 10 mg / kg for G4 (B10317, 2 mg / kg), and 0.4 mg / kg for G5 (B10317, 0.4 mg / kg). All test substances were administered intravenously to tumor-bearing mice every three days for a total of eight doses. Tumor volume and body weight were measured and recorded twice a week. The study concluded on day 22 after the last dose. On day 25 after grouping, the mean tumor volume of G1 (control group) was 1752 ± 269 mm². 3 The average tumor volume in the G2 group was 48±40 mm with a TGI of 103.3%. 3 The average tumor volume in the G3, G4, and G5 groups was 18±14 mm, with a TGI of 105.1%. 3 , 136±110mm with 97.9% TGI 3 and 470±192mm with 77.7% TGI 3The results were as follows. Average body weight and average tumor volume are shown in Tables 27 and 28. Neither BMUR nor B10317 had a negative effect on animal body weight or obvious clinical signs (Table 27). Tumor growth inhibition (TGITV) was calculated and is shown in Table 28. Both BMUR and B10317 showed considerable antitumor activity against tumor volume. The B10317 treatment group showed excellent tumor growth inhibition even at low doses, demonstrating superior efficacy against BMUR.

[0243] The mean tumor growth profile and volume at treatment are shown in Figure 15, and individual tumor volumes are shown in Figure 15. As shown in Figures 15 and 16, B10317 significantly reduced in vivo tumor growth in the MC38-hB7-H4 hoarding model in a dose-dependent manner. The BMUR-treated group (G2) also showed in vivo antitumor activity, but B10317 showed stronger in vivo efficacy (i.e., approximately 100% tumor growth inhibition) even at a 2mpk dose (G4).

[0244] [Table 27]

[0245] [Table 28]

[0246] Experimental Example 16. Confirmation of the protective effect of bispecific antibodies against re-attack of MC38-hB7-H4.

[0247] On day 22 after the final treatment, tumor cells were transplanted into 21 cured mice (7 in G2, 7 in G3, 5 in G4, and 2 in G5) and 5 naive mice in the treatment group for re-attack experiments. The mice were given B-CAG-hB7-H4 MC38 colon tumor cells (1 × 10⁶) in the entire left side for tumor development. 6 ) was administered by subcutaneous injection.

[0248] As shown in Figure 17, both BMUR and B10317 showed significant antitumor activity at the tested doses and did not negatively affect animal body weight or induce obvious clinical signs.

[0249] In the B103017 treatment groups (G3, G4, and G5), there was no tumor growth upon re-inoculation with B-CAG-hB7-H4 MC38, which promoted long-term memory protection against B-CAG-hB7-H4 MC38 tumors. Of the seven tumors, two grew slowly in the BMUR-treated cure group (G2), while the other tumors appeared early but almost disappeared later.

[0250] Overall, B10317 showed stronger long-term memory protection than BMUR for B-CAG-hB7-H4 MC38 tumors.

[0251] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0252] The scope of the present invention is defined by the claims set forth below, and all modifications or alterations derived from the meaning and scope of the claims, as well as the concept of equivalents thereof, should be construed as being included within the scope of the present invention.

Claims

1. An anti-B7-H4 / anti-4-1BB bispecific antibody comprising an anti-B7-H4 antibody or its antigen-binding fragment and an anti-4-1BB antibody or its antigen-binding fragment, The anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, including the following: (1) Heavy chain complementarity-determining region 1 (CDR-H1) of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, Light chain complementarity-determining region 1 (CDR-L1) of SEQ ID NO: 7, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; (2) CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; (3) CDR-H1 of SEQ ID NO: 25, CDR-H2 of SEQ ID NO: 26, CDR-H3 of SEQ ID NO: 27, CDR-L1 of SEQ ID NO: 29, CDR-L2 of SEQ ID NO: 30, and CDR-L3 of SEQ ID NO: 31; or (4) Including CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, CDR-H3 of SEQ ID NO: 35, CDR-L1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 38; The anti-4-1BB antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, including the following: (1) CDR-H1 of sequence number 44, CDR-H2 of sequence number 46, CDR-H3 of sequence number 48, CDR-L1 of sequence number 54, CDR-L2 of sequence number 55, and CDR-L3 of sequence number 56; (2) CDR-H1 of sequence number 44, CDR-H2 of sequence number 46, CDR-H3 of sequence number 49, CDR-L1 of sequence number 54, CDR-L2 of sequence number 55, and CDR-L3 of sequence number 56; (3) CDR-H1 of SEQ ID NO: 44, CDR-H2 of SEQ ID NO: 46, CDR-H3 of SEQ ID NO: 50, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 56; or (4) CDR-H1 of SEQ ID NO: 45, CDR-H2 of SEQ ID NO: 47, CDR-H3 of SEQ ID NO: 51, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 56; A bispecific antibody containing anti-B7-H4 / anti-4-1BB.

2. The anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 22, 24, and 32; and The anti-B7-H4 / anti-4-1BB bispecific antibody according to claim 1, comprising a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 23, 28, and 36.

3. The anti-B7-H4 antibody or its antigen-binding fragment is Sequence ID: Heavy chain framework 1 (H-FR1) containing 12 amino acid sequences; Sequence ID: Heavy chain framework 2 (H-FR2) containing 13 amino acid sequences; Sequence ID: Heavy chain framework 3 (H-FR3) containing 14 amino acid sequences; Sequence ID: Heavy chain framework 4 (H-FR4) containing 15 amino acid sequences; Sequence ID: Light chain framework 1 (L-FR1) containing 17 amino acid sequences; Sequence ID: Light chain framework 2 (L-FR2) containing 18 amino acid sequences; Sequence ID: Light chain framework 3 (L-FR3) containing 19 amino acid sequences; and The anti-B7-H4 / anti-4-1BB bispecific antibody according to claim 1 or claim 2, comprising: a light chain framework 4 (L-FR4) containing the amino acid sequence of SEQ ID NO:

20.

4. The anti-B7-H4 / anti-4-1BB bispecific antibody according to any one of claims 1 to 3, wherein each of the anti-B7-H4 antibody or its antigen-binding fragment and the anti-4-1BB antibody or its antigen-binding fragment are independently a chimeric antibody, a humanized antibody, or a human antibody.

5. An isolated polynucleotide encoding an anti-B7-H4 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 4.

6. A pharmaceutical composition for the prevention or treatment of diseases related to B7-H4, 4-1BB, or both, comprising the anti-B7-H4 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 4.

7. The pharmaceutical composition according to claim 6, wherein the disease associated with B7-H4, 4-1BB, or both is cancer.

8. The pharmaceutical composition according to claim 7, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

9. Use of an anti-B7-H4 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to any one of claims 6 to 8, for the manufacture of a pharmaceutical product for the prevention or treatment of diseases related to B7-H4, 4-1BB, or both thereof.

10. An anti-B7-H4 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, for use in the prevention or treatment of diseases related to B7-H4, 4-1BB, or both thereof.

11. Anti-B7-H4 antibodies or antigen-binding fragments thereof that are specific to the B7-H4 protein The anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, which include the following: (1) CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 7, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; (2) CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; (3) CDR-H1 of SEQ ID NO: 25, CDR-H2 of SEQ ID NO: 26, CDR-H3 of SEQ ID NO: 27, CDR-L1 of SEQ ID NO: 29, CDR-L2 of SEQ ID NO: 30, and CDR-L3 of SEQ ID NO: 31; or (4) CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, CDR-H3 of SEQ ID NO: 35, CDR-L1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 38; An anti-B7-H4 antibody or its antigen-binding fragment, including the above.

12. The anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 11, comprising the following: SEQ ID NOs: Heavy chain variable region containing an amino acid sequence selected from the group consisting of 1, 22, 24, and 32; and A light chain variable region containing an amino acid sequence selected from the group consisting of sequence numbers 5, 6, 23, 28, and 36.

13. The anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 11 or claim 12, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

14. An isolated polynucleotide that encodes an antibody or antigen-binding fragment according to any one of claims 11 to 13.

15. A pharmaceutical composition for the prevention or treatment of B7-H4 related disease, comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof according to any one of claims 11 to 13.

16. The pharmaceutical composition according to claim 15, wherein the disease associated with B7-H4 is cancer.

17. The pharmaceutical composition according to claim 16, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

18. Use of an anti-B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 11 to 14 for the manufacture of a pharmaceutical product for the prevention or treatment of a disease related to B7-H4.

19. An anti-B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 11 to 14, for use in the prevention or treatment of diseases related to B7-H4.

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