Bispecific epitope-binding proteins comprising anti-4-1BB antibodies and PD-1 proteins or fragments thereof and uses thereof

Abstract

The present invention provides a bispecific epitope binding protein comprising an anti-4-1BB agonist antibody and a PD-1 protein or a fragment thereof that binds with high affinity to PD-L1. The bispecific epitope binding protein simultaneously and independently binds to 4-1BB and PD-L1, which binds to 4-1BB-upregulated T cells, particularly effector CD8+ T cells, as well as to tumor cells expressing PD-L1. Such an interaction induces potent activation of the cytotoxic activity of anti-tumor effector CD8+ T cells by blocking PD-1 / PD-L1-mediated inhibitory signaling and activating 4-1BB-mediated costimulatory signaling in a mouse model. Such a bispecific epitope binding protein has a much more potent oncolytic effect than either component alone.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 135,812, filed January 11, 2021, the entire contents of which are incorporated herein by reference.

[0002] A reference to an array list Pursuant to 37 CFR §1.52(e)(5), this specification references a Sequence Listing that has been submitted electronically in a .txt file titled "540469WO_ST25.txt". The .txt file was created on Jan. 10, 2022 and is 120,095 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference and form a mandatory part of the disclosure of this application.

[0003] FIELD OF THE PRESENT ART The present invention provides a bispecific epitope binding protein comprising an anti-4-1BB agonist antibody and a PD-1 protein or a fragment thereof that binds with high affinity to PD-L1. The bispecific epitope binding protein simultaneously and independently binds to 4-1BB and PD-L1, which binds to 4-1BB-upregulated T cells, particularly effector CD8+ T cells, as well as to tumor cells expressing PD-L1. Such an interaction induces potent activation of the cytotoxic activity of anti-tumor effector CD8+ T cells by blocking PD-1 / PD-L1-mediated inhibitory signaling and activating 4-1BB-mediated costimulatory signaling in a mouse model. Such a bispecific epitope binding protein has a much more potent oncolytic effect than either component alone. [Background technology]

[0004] Cancer remains one of the leading causes of death in the world. According to recent statistics, 13% of the world's population will die from cancer. The International Agency for Research on Cancer (IARC) estimates that in 2020, 19.3 million new cancer cases and 10 million cancer deaths will occur worldwide. By 2030, 21.7 million new cancer cases and 13 million cancer deaths are expected worldwide due to population growth and aging and exposure to risk factors such as smoking, unhealthy diet, and physical inactivity. Additionally, pain and medical costs from cancer treatments reduce the quality of life of both cancer patients and their families. Thus, there remains a critical need to expand the arsenal available to medical professionals to address the ever-growing cancer crisis.

[0005] 4-1BB is a co-stimulatory molecule expressed on the surface of T cells and antigen-presenting cells during immune responses, and is known to be a member of the tumor necrosis factor (TNF) receptor family, which is a cell membrane protein. 4-1BB expression is rapidly induced on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. 4-1BB is a 55kDa homodimer that is expressed in various sites, such as mouse T cell lines, thymocytes, and mature T cells, and is known to be activated by ConA, phytohemagglutinin (PHA), ionomycin, anti-CD3 antibodies, or molecules. In addition, a portion of 4-1BB exists inside cells and binds to p561, a type of protein kinase, and therefore it can be inferred that extracellular signals are transmitted into cells through 4-1BB.

[0006] Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is an excellent target protein for cancer immunotherapy in the treatment of various cancers, such as malignant melanoma, non-small cell lung cancer, and renal cell carcinoma. PD-1 is a receptor present on the surface of T cells and belongs to the immunoglobulin superfamily. When PD-1 binds to programmed death ligand 1 (PD-L1) or programmed death ligand 2 (PD-L2), PD-1-associated signaling is activated, suppressing T cell proliferation, interferon gamma and interleukin 2 production, and T cell receptor signaling, thereby suppressing T cell activation. Various types of cancer cells are known to express PD-L1 to cause loss of T cell function, which acts as a mechanism for cancer cells to evade immune attack.

[0007] 4-1BB / 4-1BBL (4-1BB ligand) provides stimulatory signals, while PD-1 / PD-L1 provides inhibitory signals to T lymphocytes. Various forms of 4-1BB agonists or PD-L1 blockers show strong anti-tumor activity, mainly in regulating CD8+ T cells. Thus, synergistic or additive anti-tumor effects can be achieved by combining 4-1BB agonists with PD-L1 blockers.

[0008] Against this technical background, the inventors of the present application developed the present invention by considering the full range of therapeutic candidates, testing various combinations of antibodies and proteins, and selecting the bispecific epitope binding proteins that exhibit the best anti-tumor activity. Summary of the Invention

[0009] 4-1BB / 4-1BBL provides stimulatory signals, while PD-1 / PD-L1 provides inhibitory signals to T lymphocytes. Various forms of 4-1BB agonists or PD-L1 blockers show potent anti-tumor activity, primarily in regulating CD8+ T cells. Thus, the present inventors speculate that synergistic or additive anti-tumor effects may be achieved by combining 4-1BB agonists and PD-L1 blockers.

[0010] In view of this, the inventors of the present application have developed the present invention by considering the full range of therapeutic candidates, testing various combinations of antibodies and proteins, and selecting the bispecific epitope binding proteins that exhibit the best anti-tumor activity.

[0011] It is an object of the present invention to provide dual specific epitope binding proteins that exhibit superior anti-tumor activity.

[0012] To achieve these ends, the present invention provides bispecific epitope binding proteins comprising combinations of (VH), (VL), (X), (VH)n, (VL)n, (X)n, (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m. In the present invention, the domains may be in a single polypeptide or the "heavy chain" and "light chain" may be in separate polypeptides.

[0013] In the above, VH, VL, and / or X are linked to Y directly or through hinges and / or linkers on the N- or C-terminal sides.

[0014] In the above, VH and VL refer to the variable heavy and light chain regions, respectively, of an antibody or antigen-binding fragment thereof that binds to 4-1BB.

[0015] In the above, X is a PD-1 protein or fragment thereof that binds to PD-L1, including modified or engineered, affinity matured, and / or mutated PD-1 protein or fragment thereof.

[0016] In the above, Y refers to a fragment of a heavy chain constant region or a light chain constant region. A fragment of a heavy chain constant region may comprise one or more of the three constant region domains CH1, CH2 and CH3, and fragments thereof. A fragment of a light chain constant region may comprise the constant region domain CL or a fragment thereof. In an embodiment of the invention, Y is a fragment comprising a CH2 or CH3 domain. In an embodiment of the invention, Y is a fragment comprising a CH1 domain. In an embodiment of the invention, Y is a fragment comprising a CL domain.

[0017] In the above, n and m are each independently an integer from 1 to 5, including all integers of 1, 2, 3, 4, and 5.

[0018] The dual specific epitope binding proteins of the invention may comprise an N-terminal signal peptide. Signal peptides that may be used in the present invention are generally 12-40, 15-35, or 16-30 amino acids in length and function to prompt the cell to translocate the protein, generally to the cell membrane. The specific sequence of a signal peptide for use in the present invention is not particularly limited and may include, for example, any known signal peptide sequence. A non-limiting example of a suitable N-terminal signal peptide has the following sequence: MEWSWVFLVTLLNGIQC (SEQ ID NO:29). Although less preferred, it is also possible that the signal peptide can be located at the C-terminus.

[0019] Exemplary dual specific epitope binding proteins of the invention are SEQ ID NOs:1-10 and 14 (S01-S10). In a preferred embodiment, a dual specific epitope binding protein of the invention has the sequence of SEQ ID NO:3 (S03).

[0020] In the context of these confirmed exemplary dual specific epitope binding proteins, it is understood that sequence variations are permissible so long as the dual specific binding is preserved. Thus, the sequences of the invention possess dual specific binding affinity to 4-1BB and PD-L1 with at least about 30-40% overall sequence identity, preferably at least about 50%, 60%, 70%, 80% or 85% or more sequence identity, and more preferably at least about 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity.

[0021] In some embodiments, the dual specific epitope binding proteins of the invention are humanized antibodies.

[0022] In some embodiments are polynucleotides that express the dual specific epitope binding proteins of the invention, vectors comprising the polynucleotides, and cells comprising the polynucleotides or vectors of the invention.

[0023] Provided herein are pharmaceutical compositions comprising a dual specific epitope binding protein, any one of the nucleic acid molecules described herein, any one of the recombinant vectors described herein, or any one of the cells described herein; and a pharma- ceutically acceptable carrier.

[0024] Provided herein are kits that contain any one of the pharmaceutical compositions described herein.

[0025] Provided herein is a method for producing a bispecific epitope binding protein, the method comprising the steps of: culturing any one of the cells described herein in a culture medium under conditions sufficient to produce the bispecific epitope binding protein; and recovering the bispecific epitope binding protein from the cells and / or the culture medium.

[0026] Provided herein are methods of treating a subject in need thereof, comprising the steps of: administering to the subject a composition comprising or conveying any one of the dual specific epitope binding proteins described herein, any one of the nucleic acid molecules described herein, any one of the recombinant vectors described herein, or any one of the cells described herein to treat the disease or condition. In some embodiments, the subject has cancer or is at risk of developing cancer. In some embodiments, the cancer comprises bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, pharyngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, prostate cancer, and combinations thereof.

[0027] The above objects highlight certain aspects of the present invention. Additional objects, aspects and embodiments of the present invention are identified in the following detailed description of the invention. [Brief description of the drawings]

[0028] The present invention and many of the attendant advantages will become better understood and readily obtained by reference to the following detailed description in conjunction with the following drawings, in which: [Figure 1] Illustrates the mode of action of a dual specific epitope binding protein for dual functions of T cell engager and checkpoint inhibitor. [Diagram 2] 1 illustrates the designs for dual specific epitope binding proteins S01-S11 described in Example 1. [Diagram 3] FIG. 1 illustrates the results of SDS-PAGE analysis (see Example 2) for dual specific epitope binding proteins S01-S11 under non-reducing and reducing conditions. [Figure 4]FIG. 1 depicts the results of size-exclusion chromatography (see Example 3) for dual specific epitope binding proteins S01-S11. [Figure 5A] 1 depicts the results of surface plasmon resonance analysis of human PD1-Fc binding to PD-L1. [Figure 5B] 1 illustrates the results of surface plasmon resonance analysis of SO3 binding to PD-L1 (see Example 4). [Figure 6A] In Example 5, the analytical method for single antigen binding ELISA is illustrated. [Figure 6B] 1 illustrates results for dual specific epitope binding proteins S01-S11 binding to 4-1BB. [Figure 6C] Illustrates results for dual specific epitope binding proteins S01-S11 that bind to PD-L1. [Figure 6D] Illustrates the bispecific antibody design of the bispecific epitope binding protein S03 and null mutants S12 (4-1BB null) and S13 (PD-L1 null). [Figure 6E] Illustrated are results for the dual specific epitope binding protein S03, which binds 4-1BB, and the null mutants S12 and S13. [Figure 6F] Illustrates the results for the bispecific epitope binding protein S03, which binds PD-L1, and the null mutants S12 and S13. [Figure 7A] 1 illustrates the analytical method for the double antigen-binding ELISA of Example 6. [Figure 7B] FIG. 1 illustrates the results of a dual antigen-binding ELISA for dual specific epitope binding proteins S01-S11. [Figure 7C] Illustrates the results of a dual antigen-binding ELISA for the dual specific epitope binding protein S03, null mutants S12 (4-1BB null) and S13 (PD-L1 null). [Figure 8]Figure 8 illustrates the cell binding assay results of Example 7. Specifically, Figure 8 illustrates the expression levels of 4-1BB and PD-L1 in the various cell lines used in the assay. [Figure 9A] (MDA-MB-231-Luc) depicts the results of FACS analysis of dual specific epitope binding protein variants with PD-L1 positive cell lines. [Figure 9B] (FaDu) depicts the results of FACS analysis of dual specific epitope binding protein variants with PD-L1 positive cell lines. [Figure 9C] FIG. 1 illustrates the results of FACS analysis for dual specific epitope binding protein variants with 4-1BB positive cell lines. [Figure 9D] (MCF-7) depicts the results of FACS analysis for dual specific epitope binding protein variants with PD-L1 and 4-1BB negative cell lines (see Example 7). [Figure 10] 1 illustrates the 4-1BB bioanalysis results in Example 8 for dual specific epitope binding proteins S01-S11. [Figure 11A] 1 illustrates the 4-1BB / PD-1 combination bioassay described in Example 9. [Figure 11B] 1 illustrates the results of a 4-1BB / PD-1 combination bioanalysis on bispecific epitope binding protein S03 compared to anti-4-1BB antibody (94 kvt-engineered IgG1, SEQ ID NOs:14 and 21), anti-PD-1 antibody (pembrolizumab, MSD), anti-PD-L1 antibody (atezolizumab, Roche), and euPD-1 Fc (SEQ ID NO:22), and specific combinations of anti-4-1BB antibody and euPD-1 Fc. [Figure 12A] 1 illustrates the results of an ELISA assay testing the binding of human PD1-Fc to PD-L1 and PD-L2. [Figure 12B] Figure 1 illustrates the results of ELISA assays testing the binding of bispecific epitope binding protein S03, anti-PD-L1 antibodies, and anti-PD-L2 antibodies to PD-L1. [Figure 12C]Figure 13 illustrates the results of ELISA assays testing the binding of bispecific epitope binding protein S03, anti-PD-L1 antibodies, and anti-PD-L2 antibodies to PD-L2 (see Example 10). [Figure 13] 1 illustrates the cytotoxicity analysis results of Example 11. [Figure 14] 1 illustrates the IFN-γ analysis results of Example 12. [Figure 15A] 1 illustrates the antibody-dependent cellular cytotoxicity assay results of Example 13 against 4-1BB positive cell lines. [Figure 15B] Figure 13 illustrates the antibody-dependent cellular cytotoxicity assay results of Example 13 against PD-L1 positive cell lines. [Figure 16] 13 illustrates the complement-dependent cytotoxicity assay results of Example 14. [Figure 17A] 1 shows an analysis of the in vivo efficacy of S03, S05 and S07 using h4-1BB knock-in mice as described in Example 15. [Figure 17B] 1 illustrates the results of an in vivo efficacy study of S03, S05 and S07 using h4-1BB knock-in mice in relation to tumor size. [Figure 18A] 1 illustrates the results of an in vivo efficacy study of S03 using h4-1BB knock-in mice in relation to tumor size as described in Example 16. [Figure 18B] 1 illustrates the results of an in vivo efficacy study of S03 using h4-1BB knock-in mice on blood T cell populations. [Figure 19] Illustrates the results of an in vivo efficacy study of S03 using h4-1BB and hPD-1 double knock-in mice on tumor size as described in Example 17. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (e.g., in the fields of enzymology, biochemistry, cell biology, molecular biology, and medical sciences). Generally, the nomenclature used herein is well known and commonly used in the art.

[0030] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned in this application are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise expressly stated.

[0031] The present invention provides dual specific epitope binding proteins comprising combinations of (VH), (VL), (X), (VH)n, (VL)n, (X)n, (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m. In the present invention, the domains may be in a single polypeptide or the "heavy chain" and "light chain" may be in separate polypeptides.

[0032] In the foregoing, VH, VL, and / or X are linked to Y directly or through a hinge and / or linker on the N- or C-terminal side. For example, VH, VL, and / or X are linked to CH2 directly or through a hinge or linker, and independently, CH3 is linked to VH, VL, and / or X directly or through a hinge or linker.

[0033] In the above, VH and VL refer to the variable heavy and light chain regions, respectively, of an antibody or antigen-binding fragment thereof that binds to 4-1BB.

[0034] In the above, X is a PD-1 protein or fragment thereof that binds to PD-L1, including modified or engineered, affinity matured, and / or mutated PD-1 protein or fragment thereof.

[0035] In the above, Y refers to a fragment of a heavy chain constant region or a light chain constant region. A fragment of a heavy chain constant region may comprise one or more of the three constant region domains CH1, CH2 and CH3, and fragments thereof. A fragment of a light chain constant region may comprise the constant region domain CL. In an embodiment of the invention, Y is a fragment comprising a CH2 or CH3 domain. In an embodiment of the invention, Y is a fragment comprising a CH1 domain. In an embodiment of the invention, Y is a fragment comprising a CL domain.

[0036] In an embodiment of the invention, Y is derived from a human IgG antibody.

[0037] Heavy chain constant regions for each of IgG1 (SEQ ID NO:32), IgG2 (SEQ ID NO:34), IgG3 (SEQ ID NO:35), and IgG4 (SEQ ID NO:36), comprising the CH1, CH2, and CH3 domains in conjunction with the three constant region domains CH1, CH2, and CH3, and fragments thereof, are well known in the art. In the present invention, one or more of the three constant region domains CH1, CH2, and CH3, and fragments thereof, can be used. To that end, Y (comprising one or more constant region domains CH1, CH2, and CH3, and fragments thereof) can be derived from any of SEQ ID NOs:32, 34, 35, or 36, or a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity. In a preferred embodiment, Y (comprising one or more constant region domains CH1, CH2 and CH3, and fragments thereof) is derived from an IgG1 heavy chain constant region having a sequence of SEQ ID NO:32 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:32, wherein said variants include additions, deletions, and / or substitutions. An example of a modified heavy chain constant region is SEQ ID NO:33 from which one or more of the constant region domains CH1, CH2 and CH3, and fragments thereof for use in the present invention may be derived.

[0038] In connection with the constant region CL, and fragments thereof, the light chain constant regions for immunoglobulin kappa (SEQ ID NO:37), immunoglobulin lambda 1 (SEQ ID NO:38), and immunoglobulin lambda 2 (SEQ ID NO:39) are well known in the art. To that end, Y can be derived from any of SEQ ID NOs:37-39 or a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity. In a preferred embodiment, Y is derived from an immunoglobulin kappa light chain constant region having the sequence of SEQ ID NO:37 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:37, wherein said variants include additions, deletions, and / or substitutions.

[0039] In the above, n and m are each independently an integer from 1 to 5, including all integers of 1, 2, 3, 4, and 5.

[0040] The (VH), (VL), (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m regions, when present, may be presented in any order from N-terminus to C-terminus. Also, when multiple copies of (VH), (VL), (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m are present, they may be present in any order relative to the other regions and need not be immediately adjacent to similar types of regions.

[0041] In an embodiment of the invention, the dual specific epitope binding protein has the structure An-A'n'-B-Cm-C'm', where: A, A', C, C': VH, VL, VH-VL, VL-VH or X B:Y VH, VL, X, Y, n, and m are as defined above, and each VH, VL, X, and Y is linked directly or via a linker and / or hinge. The values ​​'n' and m' are independently integers from 1 to 5, including all integers 1, 2, 3, 4, or 5.

[0042] In an embodiment of the invention, the dual specific epitope binding protein has the structure An-B-Cm, where: A or C: VH-VL, VL-VH or X B:Y VH, VL, X, Y, n, and m are as defined above, and each VH, VL, X, and Y is linked directly or via a linker and / or hinge.

[0043] In an embodiment of the invention, the CH1 domain is C-terminally linked to (VH-VL)n, (VL-VH)n, or (X)n, either directly or via a linker and / or hinge, and the CH2 / CH3 domain is N-terminally linked to (VH-VL)m, (VL-VH)m, or (X)m, either directly or via a linker and / or hinge. Also in such an embodiment, the CH1 domain is linked to the CH2 / CH3 domain, either directly or via a linker and / or hinge, wherein VH, VL, X, n, and m are as defined above.

[0044] The present invention also includes embodiments in which the dual specific epitope binding protein has a heavy chain and a light chain, each on a separate polypeptide. In such embodiments, the heavy chain comprises VH-VL, VL-VH, X, and / or VH-CH1 linked to CH2 directly or through a linker and / or hinge, and independently, CH3 is linked to VH-VL, VL-VH, or X directly or through a linker and / or hinge. In such embodiments, the light chain comprises VL linked to CL directly or with a linker and / or hinge. VH, VL, and X are as defined above, and each VH, VL, X, CH1, CH2, CH3, and CL are linked directly or through a linker and / or hinge. As examples of such embodiments, reference is made to S09 and S10 (see FIG. 2), where S09 has a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:14, and S10 has a heavy chain of SEQ ID NO:10 and a light chain of SEQ ID NO:14.

[0045] Preferably, the dual specific epitope binding protein is in an scFv format.

[0046] In an exemplary embodiment of the invention, a dual specific epitope binding protein is provided that comprises (VH and VL, in any order): nY and / or Y-(X)m, where: Y is independently a CH2 or CH3 domain; VH, and VL, and CH2 and / or CH3, or CH2 and / or CH3 and X, are linked directly or via a hinge and / or linker; VH and VL refer to the variable heavy and light chain regions, respectively, of an antibody or antigen-binding fragment thereof that binds to 4-1BB; X is a PD-1 protein or fragment thereof that binds to PD-L1, including a modified or engineered, affinity matured, and / or mutated PD-1 protein or fragment thereof; n and m are each an integer from 1 to 5, including all integers of 1, 2, 3, 4, or 5.

[0047] A specific example of such an embodiment is an scFv arranged as follows, from N-terminus to C-terminus: (VL-VH)n-CH2-CH3-(X)m. VL, VH, X, n, and m are as defined above. In such an example embodiment, VL and VH are linked by a linker, VH and CH2 are linked by a linker, CH3 and (X)m are / are linked by a linker, or CH2 and CH3 are linked by a linker. In such an embodiment, one or more of the linkers are followed or followed by a hinge. Alternatively, one or more of the aforementioned links can be directly linked or linked by a hinge.

[0048] Yet another exemplary embodiment of the invention is a dual specific epitope binding protein comprising (X)Y and / or Y-(VH and VL, in any order), where: Y is independently a CH2 or CH3 domain; VH, and VL, and CH2 and / or CH3, or CH2 and / or CH3 and X, are linked directly or through a hinge and / or linker; VH and VL refer to the variable heavy and light chain regions, respectively, of an antibody or antigen-binding fragment thereof that binds to 4-1BB; X is a PD-1 protein or fragment thereof that binds to PD-L1, including modified or engineered, affinity matured, and / or mutated PD-1 protein or fragment thereof; n and m are each an integer from 1 to 5, including all integers of 1, 2, 3, 4, or 5.

[0049] A particular example of this embodiment is an scFv arranged as follows, written from N-terminus to C-terminus: (X)m-CH2-CH3-(VH-VL)n. VL, VH, X, n, and m are as defined above. In such example embodiments, (X)m and CH2 are linked by a linker, CH3 and VH are linked by a linker, VH and VL are / are linked by a linker, or CH2 and CH3 are linked by a linker. In such embodiments, one or more of the linkers are followed by or are followed by a hinge. Alternatively, one or more of the aforementioned links may be direct or by a hinge.

[0050] In an embodiment of the invention, each of n and m is independently selected from the integers 1, 2, 3, 4, or 5, and any combination of n and m having such integer values ​​can be present. When multiple VH, VL, and / or X are present (i.e., n and / or m are greater than 1), they can be directly linked or the copies can be separated by hinge and / or linker sequences. In a preferred embodiment, the multiple copies are separated by linker sequences.

[0051] A bispecific epitope binding protein comprising (VH and VL, in any order)nY and / or Y-(X)m according to the invention is a first arm of a bispecific therapeutic agent and is paired with a second arm of a bispecific epitope binding protein comprising the same (VH and VL, in any order)nY and / or Y-(X)m. If the first arm of the pair is (X)mY and / or Y-(VH and VL, in any order)n, then similarly the second arm of the bispecific epitope binding protein will comprise the same (X)mY and / or Y-(VH and VL, in any order)n. That is, the generated antibody constituting the bispecific epitope binding protein of the invention must have the same sequence in one arm as the other arm of the pair.

[0052] VH, and VL and CH2 and / or CH3, or CH2 and / or CH3 and X, can be linked directly or through a hinge and / or linker. Cysteine ​​residues can be included in the linking parts for linkage through a hinge and / or linker. VH, VL and CH2 and / or CH3, or CH2 and / or CH3 and X, can be linked directly or through a hinge and / or linker, while the cysteine ​​residues form disulfide bonds.

[0053] Considering all sizes of therapeutic candidates and various combinations of antibodies and proteins, the best anti-tumor activity was demonstrated through the combination of an anti-4-1BB antibody fragment and a soluble PD-1 that binds with high affinity to PD-1.

[0054] In an embodiment of the invention, the VH region comprises SEQ ID NO: 23. In an embodiment of the invention, the VL region comprises SEQ ID NO: 25. In an embodiment of the invention, the anti-4-1BB antibody fragment has a VH region comprising SEQ ID NO: 23 and a VL region comprising SEQ ID NO: 25.

[0055] In an embodiment of the invention, the anti-4-1BB antibody fragment is in scFv format.

[0056] In an embodiment of the invention, the nucleic acid encoding SEQ ID NO:23 is the sequence of SEQ ID NO:24. In an embodiment of the invention, the nucleic acid encoding SEQ ID NO:25 is the sequence of SEQ ID NO:26.

[0057] SEQ ID NO: 23: QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSS

[0058] SEQ ID NO: 24: caggtccagctggtgcagagcggcgccgaagtgaaaaaacctggggcaagtgtcaagctgtcctgtaaggccagcggttataccttctcctcatattggatgcactgggtgaggcaagcccctggacaagggctggaatggatcggtgaaattaatcccggaaatggccatacaaactacaatgaaaaattcaaaagtcgagtgaccatgacacgggacacatccacttccactgcatacatggagctttcgagtctgcgctccgaggatacagctgtctattactgcgcacgcagttttaaaactgccagagcctttgcctactggggtcagggaaccctggtcaccgttagcagc

[0059] SEQ ID NO: 25: DIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIK

[0060] SEQ ID NO: 26: gacattgtgatgacacagtcccctgctttcctgagcgttacacccggcgaaaaggtgactatcacatgcagggctagtcagaccatctcagactaccttcattggtatcaacagaagccagaccaggctcctaagttgctgataaagtacgcctcccaatccatttccggcattccttcccgtttttccggctccggctccggcaccgactttacgttcaccatctcttctttggaggctgaagacgcagctacctattactgtcaggatggtcacagctggccaccaactttcgggcaaggcaccaagctggagatcaaa

[0061] The PD-1 protein or fragment can be used with / without affinity maturation, so long as it binds to PD-L1. Thus, the PD-1 protein or fragment can be a native PD-1 or fragment. Alternatively, the PD-1 protein or fragment can be modified or engineered and affinity matured / mutated. To obtain a molecularly evolved PD-1, a 3D complex between PD-1 and PD-L1 was first used to select major contributing PD-1 amino acids, random mutations were constructed at the selected amino acids, and yeast surface display was used for screening. The affinity matured PD-1 was designated euPD-1. The PD-1 protein or fragment thereof retains specific binding affinity for PD-L1 in conjunction with a mutation in the PD-1 protein or fragment thereof having at least about 30-40% overall sequence identity, preferably at least about 50%, 60%, 70%, 80%, or 85% or more sequence identity, and more preferably at least about 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity.

[0062] In an embodiment of the invention, euPD-1 may comprise the sequence of SEQ ID NO: 27. Consistent with the foregoing, a PD-1 protein or fragment thereof may have at least about 30-40% overall sequence identity to SEQ ID NO: 27, preferably at least about 50%, 60%, 70%, 80%, or 85% or more sequence identity to SEQ ID NO: 27, and more preferably at least about 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27, wherein the PD-1 protein or fragment thereof retains specific binding affinity for PD-L1.

[0063] In an embodiment of the invention, the nucleic acid encoding SEQ ID NO:27 is the sequence of SEQ ID NO:28.

[0064] SEQ ID NO: 27: FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFHMSVVRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG

[0065] SEQ ID NO: 28: tttctcgaatcaccggacagaccctggaatgcgcccacattctcaccagcacttttgctggtagcagagggcgataatgctacattcacgtgttccttcagtaatgcaagcgagtcatttcatgtggtttggcatcgagagtcacctagtgggcagactgatacacttgccgcattcccggaagatcgctcccagccaggtcaggatcaccggttcagggtaacccgactgccgaatgggcgcgatttccatatgagcgttgtccgggcgcaacggaacgatagtggaacatacgtgtgtggcgtaatatccctcgctcccaaaatacaaataaaggagtctctgagagcagagctgagagtgacagaacgacgggcggaagttcccacggctcatccgtcaccaagtccgcgccccgcaggccaatttcaaacgctcgtcgtaggc

[0066] The dual specific epitope binding proteins of the invention can include a signal peptide, e.g., an N-terminal signal peptide. Signal peptides that can be used in the invention are generally 12-40, 15-35, or 16-30 amino acids in length and function to promote the translocation of a protein to the cell, generally to the cell membrane. The specific sequence of a signal peptide for us in the present invention is not particularly limited and can include, for example, any known signal peptide sequence. A non-limiting example of a suitable N-terminal signal peptide has the following sequence: MEWSWVFLVTLLNGIQC (SEQ ID NO:29). It is also possible, although less preferred, that the signal peptide can be located at the C-terminus.

[0067] Exemplary dual specific epitope binding proteins of the invention are SEQ ID NOs:1-10 and 14 (S01-S10). In a preferred embodiment, a dual specific epitope binding protein of the invention has the sequence of SEQ ID NO:3 (S03).

[0068] In connection with such confirmed exemplary dual specific epitope binding proteins, it is understood that sequence variations are permissible so long as the dual specific binding is preserved. Thus, sequences of the invention that possess dual specific binding affinity for 4-1BB and PD-L1 have at least about 30-40% overall sequence identity, preferably at least about 50%, 60%, 70%, 80%, or 85% or more sequence identity, and more preferably at least about 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity.

[0069] Through cell binding and dual antigen analysis, it was confirmed that S03 (SEQ ID NO:3) binds to two targets simultaneously and independently.

[0070] Consistent with the 4-1BB bioassay, biologically relevant MOA-based assays demonstrated potent PD-L1-dependent T cell activation by S03.

[0071] When tested against human PD-L1-expressing tumor cells in a 4-1BB knock-in mouse model, S03 demonstrated a much more potent tumor lytic effect than each component alone and other candidates such as S05 or S07.

[0072] Moreover, when S03 was infused, it increased T cells, especially effector CD8+ T cells, in the peripheral blood, and consequently reduced the size of the tumor.S03 is a promising anti-tumor agent that promotes CD8+ T cell infiltration and activates T cells in situ at the tumor site by simultaneously binding to two different targets, 4-1BB and PD-L1.

[0073] As a result, S03 is a dual-specific epitope binding protein comprising anti-4-1BB antibody and euPD-1, and exhibits the following properties: 1. Reduced ADCC and CDC activity through Fc engineering; 2. Stronger binding affinity than native PD-1; 3. Selectively activate and enhance PD-L1-benign tumor-indicative T-cell responses in in vitro preclinical studies. 4. Better tumor eradication effect than the control antibody.

[0074] "Bispecific" or "dual specific" refers to a property of a binding protein that can specifically bind to targets and modulate the activity of two different targets, the production being accomplished by an antibody or protein that specifically binds to each target, or fragment thereof, and possesses two distinct antigen-binding epitopes that are monovalent for each antigen that binds thereto (specificity for two targets). In particular, the bispecific epitope binding proteins of the invention relate to having specific binding of or specific affinity for 4-1BB and PD-L1.

[0075] As used herein, the term "antibody" refers to an anti-4-1BB antibody that specifically binds to 4-1BB. The scope of the present invention includes not only intact antibody forms that specifically bind to 4-1BB, but also antigen-binding fragments of the antibody molecule.

[0076] The term "PD-1 protein" includes PD-1 protein or fragments that bind to PD-L1. The scope of the present invention includes PD-1-binding fragments of recombinant protein molecules as well as intact recombinant protein forms that specifically bind to PD-L1. Also, as noted above, the PD-1 protein or fragment can be used with or without affinity maturation, so long as it binds to PD-L1. Thus, the PD-1 protein or fragment can be a native PD-1 or a fragment thereof. Alternatively, the PD-1 protein or fragment thereof can be modified or engineered, affinity matured, and / or mutated consistent with the above description.

[0077] Intact antibodies have a structure with two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond.

[0078] As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH with sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3, and fragments thereof. As used herein, the term "light chain" refers to a full-length light chain comprising a variable region domain VL with sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL, and fragments thereof.

[0079] Whole antibodies include IgA, IgD, IgE, IgM and IgG subtypes or variants, in particular IgG includes IgG1, IgG2, IgG3 and IgG4. Heavy chain constant regions include gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subtypes gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). Light chain constant regions include kappa (κ) and lambda (λ) types.

[0080] Antigen-binding fragments of antibodies or antibody fragments refer to fragments having antigen-binding function, and include Fab, F(ab'), F(ab')2, Fv, etc. Among antibody fragments, Fab has a structure having light and heavy chain variable regions, a light chain constant region and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 fragments are formed when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0081] Fv corresponds to the minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In a dual-chain Fv (2-chain Fv), the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are linked by a covalent bond through a peptide linker or directly from the C-terminus to form the same structure as a dimer as the dual-chain Fv. Such antibody fragments can be produced by proteolytic enzymes (for example, limited digestion of a complete antibody with papain to produce Fab, which is then digested with pepsin to produce F(ab')2), or by genetic recombination techniques.

[0082] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. Such a region is a dimer of one heavy chain variable domain and one light chain variable domain.

[0083] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments contain a pair of Fab' fragments covalently linked by cysteines in a hinge region generally located at the C-terminus of the Fab' fragments.

[0084] "Single-chain Fv (scFv)" antibody fragments are constructs made up of a single polypeptide chain containing the VH and VL domains of an antibody. A polypeptide linker can additionally be included between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0085] In the present invention, the term "hinge" is not particularly limited and is well known in the art. Hinge refers to a flexible stretch of amino acids in the central part of the heavy chain of the IgG and IgA immunoglobulin classes, which connects these two chains by disulfide bonds. It is rich in cysteine ​​and proline amino acids, is highly variable in amino acid sequence, and is not similar to any other immunoglobulin region. The hinge region may vary in length, generally ranging from 10 to 50 amino acid residues. See the review in the literature [Adlersberg JB, Ric Clin Lab. Jul-Sep 1976; 6(3): 191-205]. As an example, reference is made to the hinge sequences EPKSCDKTHTCPPCP (SEQ ID NO: 30) and DKTHTCPPCP (SEQ ID NO: 31).

[0086] It is particularly advantageous in the present invention to have the hinge followed by a linker sequence, which may also be preceded by the hinge.

[0087] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFvs, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of antibodies.

[0088] The heavy chain constant region can be selected from any one of the following isotypes: gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region can be of the kappa or lambda type.

[0089] A monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., identical except for naturally occurring mutations that may be present in small numbers of antibodies. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Unlike conventional (polyclonal) antibodies, which generally contain different antibodies directed against different antigenic groups (epitopes), each monoclonal antibody is directed against a single antigenic group.

[0090] The term "epitope" refers to a group of protein crystals to which an antibody can specifically bind. Epitopes generally consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0091] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the hypervariable regions of the recipient are replaced by residues from the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that retain the desired specificity, affinity, and capacity.

[0092] The term "human antibody" refers to a molecule derived from human immunoglobulin in which the entire amino acid sequence that constitutes the antibody, including the complementarity determining regions and structural regions, is composed of human immunoglobulin.

[0093] Chimeric antibodies (immunoglobulins) and chemical antibodies, which are fragments of antibodies exhibiting the desired biological activity, have portions of their heavy and / or light chains that are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies from other species or belonging to other antibody classes or subclasses.

[0094] The "variable region" of an antibody as used herein refers to the light and heavy chain portions of an antibody molecule that contain the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0095] The term "complementarity determining region (CDR)" refers to the amino acid residues of an antibody variable domain that are responsible for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

[0096] "Framework regions (FR)" are those variable domain residues other than the CDR residues. Each variable domain generally has four FRs: FR1, FR2, FR3 and FR4.

[0097] An scFv is an antibody fragment that is a construct consisting of a single polypeptide chain containing the VH and VL domains of an antibody, and may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0098] In particularly preferred embodiments of the invention, the dual specific epitope binding protein is a single polypeptide.

[0099] The linker may be a peptide linker and may have a length of about 10-25 aa, about 10-20 aa, or about 12-19 aa, including each integer value as recited, and may include, but is not limited to, hydrophilic amino acids such as, for example, glycine and / or serine.

[0100] In particular, the linker may be, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10, i.e., n and m can be independently any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), but the linker can be, for example, (G n S) m (n and m each range from 1 to 10, i.e., n and m may independently be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Specific reference is given to the (G4S)3 (SEQ ID NO:15) or (G4S)2 (SEQ ID NO:16) linkers. The linker may also be one or more copies of the 218 linker (SEQ ID NO:17) or the 218S linker (SEQ ID NO:18). Of course, combinations of the above-mentioned linkers are also contemplated in the present invention.

[0101] In an embodiment, the present invention relates to nucleic acids encoding dual specific epitope binding proteins. Dual specific epitope binding proteins can be recombinantly produced by isolating the nucleic acid. Thus, the present invention includes nucleic acids encoding dual specific epitope binding proteins, vectors comprising nucleic acids encoding dual specific epitope binding proteins, and cells comprising the nucleic acids and / or vectors.

[0102] "Nucleic acid" is interpreted to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, are interpreted to include not only natural nucleotides but also analogs in which the sugar or base site is modified. The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0103] The DNA can be easily isolated or synthesized using existing molecular biology techniques, and the nucleic acid can be isolated and inserted into a replicable vector for further cloning (DNA amplification) or further expression. Based on this, the present invention relates to a recombinant expression vector comprising the nucleic acid of another embodiment.

[0104] As used herein, the term "vector" includes plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and the like, as a means for expressing a target gene in a host cell. Components of a vector typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the dual specific epitope binding protein is operably linked to a promoter, a transcription termination sequence, and the like.

[0105] The term "operably linked" refers to a functional link between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional modulator binding sites) and another nucleic acid sequence, where the control sequence also controls the transcription and / or translation of the other nucleic acid sequence.

[0106] When a prokaryotic cell is the host, it generally contains a strong promoter capable of propagating transcription (e.g., the tac promoter, the lac promoter, the lacUV5 promoter, the lpp promoter, the pLλ promoter, the pRλ promoter, the rac5 promoter, the amp promoter, the recA promoter, the SP6 promoter, the trp promoter and the T7 promoter, etc.), a ribosome binding site for translation initiation, and transcription / translation termination sequences. Also, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally includes a polyadenylation sequence as a transcription termination sequence.

[0107] In some circumstances, the vectors can be fused to other sequences to facilitate purification of the bispecific epitope binding proteins expressed therefrom, including, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6x His (hexahistidine; Qiagen, USA), etc.

[0108] The vectors contain antibiotic resistance genes commonly used in the art as selectable markers, for example, genes conferring resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0109] In yet another aspect, the invention relates to a host cell transfected with a recombinant expression vector. The host cells used to produce the dual specific epitope binding proteins of the invention can be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

[0110] Prokaryotic host cells such as strains of Bacillus, including Escherichia coli, Bacillus subtilus, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0111] However, animal cells are of most interest and examples of useful host cell lines may be, but are not limited to, MDA-MB-231, MCF-7, FaDu, Jurkat, MC38, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0112] In yet another aspect, the present invention relates to a method for producing a dual specific epitope binding protein comprising the steps of: culturing a host cell to produce the dual specific epitope binding protein; and isolating and purifying the produced dual specific epitope binding protein.

[0113] The host cells can be cultured in various media. Any commercially available medium can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art can be included at appropriate concentrations. The culture conditions such as temperature, pH, etc. should already be used for the host cell selected for expression and be obvious to those skilled in the art.

[0114] To recover the bispecific epitope binding protein, impurities can be removed through centrifugation or ultrafiltration, and the resultant can be purified using methods similar to affinity chromatography. Other additional purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc.

[0115] In yet another aspect, the invention relates to a composition for preventing or treating tumors or cancer comprising a dual specific epitope binding protein.

[0116] The term "prevention" does not require absolute elimination of breakthrough cases. Thus, the term "prevention" refers to any action of suppressing or slowing the progression of clinical symptoms of a disease by administration of a composition according to the invention, and the term "treatment" refers to suppressing the development of clinical symptoms of a disease or alleviating or eliminating clinical symptoms.

[0117] The pharma- ceutically acceptable carriers contained in the composition of the present invention are commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The composition of the present invention may further include, in addition to the above-mentioned components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0118] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration.

[0119] Since proteins or peptides are digested when administered orally, oral compositions must be formulated to coat or protect the active agent from degradation in the stomach, and the pharmaceutical composition can be administered by any device capable of delivering the active agent to the target cells.

[0120] The appropriate dosage of the composition according to the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, medical condition, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient. A skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg, where "kg" refers to the body weight of the subject. The lower limit of the daily dosage may be 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, and 40 mg / kg. The upper limit of the daily dose may be 100, 97.5, 95, 92.5, 90, 87.5, 85, 82.5, 80, 77.5, 75, 72.5, 70, 67.5, 65, 62.5, 60, 57.5, 55, 52.5, 50, 47.5, 45, 42.5, and 40 mg / kg. Also included by the present invention are any ranges defined by lower and upper limits selected from the above, and ranges in which the value mentioned as the lower limit can act as an upper limit (e.g., a range of 0.01-10 mg / kg is included by the present invention) and ranges in which the value mentioned as the upper limit can act as a lower limit (e.g., a range of 45-75 mg / kg is included by the present invention). As used herein, the term "pharmacologically effective dose" refers to a dose sufficient to prevent or treat cancer.

[0121] The pharmaceutical composition of the present invention can be formulated using pharma- ceutically acceptable carriers and / or excipients to be prepared in unit doses, or can be prepared in multi-dose containers by a method that can be easily carried out by a person skilled in the art to which the present invention pertains. In this case, the formulation can be in the form of a solution in an oil or aqueous medium, a suspension or an emulsion, or in the form of an extract, powder, suppository, granule, tablet or capsule. Dispersants or stabilizers can be additionally included.

[0122] In some embodiments, the bispecific epitope binding proteins, single-chain chimeric polypeptides, or nucleic acid constructs described herein can be used to treat a subject in need thereof.

[0123] In some embodiments, a pharmaceutical composition comprising a bispecific epitope binding protein, a single-chain chimeric polypeptide and a pharma- ceutically acceptable carrier can be administered to a subject diagnosed with a disease or condition.

[0124] In some embodiments, the subject has cancer or is at risk of developing cancer.

[0125] In some embodiments, the pharmaceutical composition may be administered with one or more additional anti-tumor therapies, including, but not limited to, ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors.

[0126] Cancer can refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can lead to the formation of malignant tumors that invade surrounding tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer or cancerous tissue can include tumors.

[0127] Cancers suitable for treatment with the methods of this disclosure include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, pharyngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, cancers for treatment with the methods of this disclosure include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In some embodiments, the cancer can include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0128] Also provided herein are compositions (e.g., pharmaceutical compositions) comprising at least one of any of the bispecific epitope binding proteins, single-chain chimeric polypeptides, any cells, or any nucleic acids described herein. In some embodiments, a composition comprises at least one of any of the bispecific epitope binding protein single-chain chimeric polypeptides described herein. In some embodiments, a composition comprises any immune cell (e.g., any immune cell described herein, e.g., any immune cell produced using any of the methods described herein).

[0129] In some embodiments, the pharmaceutical composition is formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical composition can include a pharma- ceutically acceptable carrier (e.g., phosphate buffered saline).

[0130] Single or multiple administrations of the pharmaceutical composition can be provided to a subject in need thereof, for example, at a dosage and frequency as required and tolerated by the subject. The dosage form should provide a sufficient amount of the active agent to effectively treat, prevent, or ameliorate a condition, disease, or symptom.

[0131] Also provided herein are kits that include any of the bispecific epitope binding proteins, single-chain chimeric polypeptides, compositions, nucleic acids, or cells (e.g., immune cells) described herein. In some embodiments, the kits can include instructions for carrying out any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

[0132] Definition: about: The term "about" when used in reference to a value herein refers to a similar value in relation to the stated value. Generally, a person of ordinary skill in the art familiar with the context will understand the appropriate degree of variation encompassed by "about" in that context. For example, in some embodiments, the term "medicament" can include a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated value.

[0133] Dosage:As used herein, the term "administration" typically refers to administering a composition to a subject or system to achieve delivery of the composition or formulation contained therein. One of skill in the art will recognize various routes that may be used to administer to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (which may be or include one or more localized sites, e.g., dermal, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may include only a single dose. In some embodiments, administration can involve the application of a fixed number of doses. In some embodiments, administration can include administration that is intermittent (e.g., multiple doses separated by time) and / or periodic (e.g., individual doses separated by a common time period) administration. In some embodiments, administration can include continuous administration (e.g., perfusion) for at least a selected time period.

[0134] Affinity: "Affinity," as known in the art, is a measure of how strongly a particular ligand binds to its partner. Affinity can be measured in a variety of ways. In some such embodiments, affinity is measured by a quantitative assay. In some such embodiments, the binding partner concentration can be fixed in excess of the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some such embodiments, the binding partner concentration and / or the ligand concentration can be varied. In some such embodiments, affinity can be compared to a standard at comparable conditions (e.g., concentration).

[0135] Antibody preparations: As used herein, the term "antibody preparation" refers to a preparation that specifically binds to a particular antigen. In some embodiments, the term includes any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody preparations include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, the antibody preparation may include one or more sequence elements, such as humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody preparation" is used to refer to one or more of the constructs or formats known or developed in the art to utilize the structural and functional characteristics of antibodies in alternative presentations. For example, in embodiments, antibody formulations used in accordance with the invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, e.g., IgNAR or fragments thereof); cameloid antibodies; shielding antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs"). TM ": S mall M odd I mmuno Pharmaceuticals; single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody formulation may lack covalent modifications (e.g., glycan attachments) that it has when produced in nature. In some embodiments, the antibody formulations can include covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., poly-ethylene glycol, etc.). In many embodiments, the antibody formulations are or include polypeptides whose amino acid sequences include one or more structural elements recognized by those of skill in the art as complementarity determining regions (CDRs); in some embodiments, the antibody formulations are or include polypeptides whose amino acid sequences include at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are identical in sequence or include 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109, 110, 111, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 Substantially identical to the reference CDR in that it exhibits 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs is deleted, added, or substituted compared to the reference CDRs, but the included CDRs have the same amino acid sequence, unlike the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids in the included CDRs are deleted, added, or substituted compared to the reference CDRs, but the included CDRs have the same amino acid sequence, unlike the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs is substituted compared to the reference CDRs, but the included CDRs have the same amino acid sequence, unlike the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids within the included CDRs are deleted, added, or substituted compared to the reference CDRs, but the included CDRs differ from the reference CDRs and have identical amino acid sequences. In some embodiments, the antibody preparation is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody preparation is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, the antibody preparation is or comprises at least a portion of a chimeric antigen receptor (CAR).

[0136] antigen:As used herein, the term "antigen" refers to a preparation that binds to an antibody preparation. In some embodiments, an antigen binds to an antibody preparation and may or may not induce a specific physiological response in an organism. In general, an antigen can be or include any chemical entity, such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (biological polymers [e.g., nucleic acid and / or amino acid polymers] and non-biological polymers [e.g., polymers other than nucleic acid or amino acid polymers]), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a glycan. Those skilled in the art will generally understand that an antigen may be provided in an isolated or pure form, or alternatively, may be provided in a crude form (e.g., together with other substances, e.g., in an extract such as a cell extract or other relatively crude preparation of an antigen-containing source). In some specific embodiments, the antigen is present in a cellular context (e.g., the antigen is expressed on the surface of a cell or is expressed in a cell). In some embodiments, the antigen is a recombinant antigen.

[0137] Antigen-binding domain: As used herein, refers to an antibody preparation or a portion thereof that specifically binds to a targeting moiety or entity. Typically, the interaction between the antigen-binding domain and its target is non-covalent. In some embodiments, the targeting moiety or entity may be of any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain may be or include a polypeptide (or a complex thereof). In some embodiments, the antigen-binding domain is part of a fusion polypeptide.

[0138] Related to:Two events or entities are "associated" with each other, as that term is used herein, when the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, genetic trait, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are in / held in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0139] Join: As used herein, the term "binding" will be understood to generally refer to a non-covalent bond between or among two or more entities. "Direct" binding includes physical contact between the entities or moieties; indirect binding includes physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts, including when the interacting entities or moieties are typically studied individually, or in the context of more complex systems (e.g., while covalently or otherwise bound to carrier entities and / or biological systems or cells).

[0140] cancer:The terms "cancer," "malignant," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, unregulated or autonomous growth and an abnormal growth phenotype characterized by a significant loss of cell proliferation control. In some embodiments, a tumor may be or include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. This disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the associated cancers may be characterized as solid tumors. In some embodiments, the associated cancers may be characterized as hematological tumors. In general, examples of various types of cancer known in the art include blood cancers including, for example, leukemia, lymphoma (Hodgkin's and non-Hodgkin's), myeloma and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissues, squamous cell carcinoma of the oral cavity, throat, pharynx, and lung, liver cancer, genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancer, benign lesions such as papilloma, etc.

[0141] Chemotherapy agents:The term "chemotherapeutic agent" as used herein has its art-recognized meaning to refer to one or more proapoptotic, cytostatic and / or cytotoxic agents, and specifically includes, for example, agents that are used and / or recommended for treating one or more diseases, disorders or conditions associated with unwanted cell proliferation. In many embodiments, the chemotherapeutic agent is useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent is one or more alkylating agents (e.g., bifunctional alkylating agents, such as Cyclophosphamide, Mechlorethamine, Chlorambucil, and Melphalan, and monofunctional alkylating agents, such as Dacarbazine, Nitrosoureas, Temozolomide), one or more anthracyclines (e.g., Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, and Mitoxantrone), Valrubicin, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents, The chemotherapeutic agent may be or may include, for example, taxanes, maytansines and their analogs, specific examples include Paclitaxel, Docetaxel, Arabaxane, and Taxotere), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide proglobulin analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., share related anti-proliferative activity):One or more of the following drugs may be used: Actinomycin, All-trans retinoic acid, Auristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin in), Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin Idarubicin, Imatinib, Irinotecan, Maytansine and / or their analogues (e.g., DM1), Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Maytansinoids, Oxaliplatin In some embodiments, the chemotherapeutic agent may be or include: Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof.It can be utilized in the context of antibody-drug conjugates. In some embodiments, the chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADCs, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine.

[0142] Operated:Generally, the term "engineered" refers to a manner in which something has been manipulated by the hand of man. For example, a polypeptide is considered to be "engineered" when the polypeptide sequence has been manipulated by the hand of man. For example, in some embodiments of the invention, an engineered polypeptide includes a sequence that includes one or more amino acid mutations, deletions, and / or insertions introduced by the hand of man in a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been fused (i.e., covalently linked) by the hand of man to one or more additional polypeptides to form a fusion polypeptide that does not naturally occur in vivo. Similarly, a cell or organism is considered to be "engineered" when it has been engineered such that its genetic information has been altered (e.g., new genetic material not previously present is introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, e.g., by substitution or deletion mutations, or mating protocols). As is common practice and understood by those of skill in the art, derivatives and / or progeny of engineered polypeptides or cells will generally still be referred to as "engineered," even though in fact the engineering was previously performed in an individual.

[0143] Pharmaceutical Composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharma- ceutical acceptable carriers. In some embodiments, the composition is adapted for administration to a human or animal subject. In some embodiments, the active agent is present in a unit dosage suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0144] Pharmaceutically acceptable carriers:In addition to the examples given above, as used herein, the term "pharmaceutically acceptable carrier" has its art-recognized meaning of generally referring to a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful within the present invention into or to a patient so that it can perform its intended function. Typically, such compositions are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other components of the dosage form, including the compound useful within the present invention, and not harmful to the patient. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compound useful within the present invention and are physiologically acceptable to the patient. The term "pharmaceutically acceptable carrier" can further include pharmaceutically acceptable salts of the compound useful within the present invention. Other additional components that can be included in pharmaceutical compositions used in the practice of the invention are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference. A "pharmaceutically acceptable carrier" is generally useful in the preparation of a pharmaceutical composition that is compatible with the other components of the composition, is not harmful to the recipient, and is not biologically or otherwise objectionable. A "pharmaceutically acceptable carrier" includes one or more carriers. Embodiments include carriers for topical, ocular, parenteral, intravenous, intraperitoneal, intramuscular, sublingual, nasal, or oral administration. A "pharmaceutically acceptable carrier" also includes formulations for the preparation of aqueous dispersions and sterile powders for injection or dispersion.

[0145] Pharmaceutically acceptable salts:As used herein, the term "pharmaceutical acceptable salts" has its commonly accepted meaning in the art and refers to derivatives of the compounds provided herein that have been modified by converting an acid or base moiety present in the parent compound into its salt form. Examples of pharmaceutical acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutical acceptable salts of the compounds provided herein include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutical acceptable salts of the compounds provided herein can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by combining the free acid or base form of such compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

[0146] Excipients: As used herein, the term "excipient" has its generally art-recognized meaning and refers to a physiologically compatible additive useful in the preparation of a pharmaceutical composition. Examples of pharma- ceutically acceptable carriers and excipients can be found, for example, in Remington Pharmaceutical Science, 16th Ed.

[0147] Polypeptides:As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art will appreciate that the term "polypeptide" is intended to include not only polypeptides having the complete sequences cited herein, but also generally enough to include polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that retain one or more activities). Moreover, those of skill in the art will appreciate that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often at least about 50%, 60%, 70%, 80%, or 85% or more, and more typically includes at least one region of much higher identity in one or more highly conserved regions, often at least about 90%, 95%, 96%, 97%, 98% or 99% or more, and generally includes at least 3-4 and often up to 20 or more amino acids, together with other polypeptides of the same genus, is included in the related term "polypeptide" as used herein. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins can contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids in length. In some embodiments, the protein is an antibody preparation, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0148] Recombination:As used herein, it is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, produced, manufactured and / or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant, combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that has been transformed or otherwise engineered to express a gene or gene or genetic component that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements or domains thereof; and / or a polypeptide that is manufactured, expressed, produced or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing the selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements arise from mutagenesis (e.g., in vivo or in vitro) of a known sequence element from a natural or synthetic source, such as the germline of a source organism of interest (e.g., human, mouse, etc.).

[0149] Specific binding:As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the environment in which binding occurs. A binding agent that interacts with one particular target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete for an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.

[0150] Subjects: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is vulnerable to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics that are characteristic of a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom or to whom a diagnosis and / or therapy is / are administered.

[0151] Treatment:As used herein, the phrase "therapeutic agent" generally refers to any formulation that induces a desired pharmacological effect when administered to an organism. In some embodiments, a formulation is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, the suitable population may be a population of model organisms. In some embodiments, the suitable population may be defined by various criteria, such as a particular age group, sex, genetic background, pre-existing clinical conditions, and the like. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is a formulation that has been approved or must receive approval by a government agency before it can be sold for administration to humans. In some embodiments, a "therapeutic agent" is a formulation that requires a medical prescription for administration to humans.

[0152] Therapeutically Effective Amount:As used herein, the term "therapeutically effective amount" refers to an amount sufficient when administered to a population suffering from or susceptible to a disease, disorder, and / or condition by a therapeutic administration regimen to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, stabilizes / delays the onset of, one or more characteristics of, a disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. For example, in some embodiments, the term "therapeutically effective amount" in the context of the therapy of the present invention refers to an amount that, when administered to an individual in need thereof, blocks, stabilizes, attenuates, or reverses cancer-supportive processes occurring in said individual, or that enhances or increases cancer-suppressive processes in said individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, prevents, stabilizes, inhibits, or reduces the further development of cancer in the individual. Particularly preferred "therapeutically effective amounts" of the compositions described herein serve to reverse the onset of, or achieve or prolong the remission of, a malignant tumor, such as pancreatic carcinoma (in a therapeutic treatment). The therapeutically effective amount administered to an individual to treat cancer may be the same or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer treatments, the therapeutic methods described herein should not be analyzed or limited to, or otherwise limited as, a "treatment" for cancer; rather, the therapeutic methods relate to the use of the compositions described to "treat" cancer, i.e., to effect favorable or beneficial changes in the health of an individual with cancer.Such benefits are recognized by skilled medical providers in the field of oncology and include, but are not limited to, stabilization of the patient's condition, reduction in tumor size (tumor regression), improved vital function (e.g., improved function of cancerous tissue or organs), reduction or inhibition of additional metastases, reduction in opportunistic infections, increased survival rates, reduction in pain, improved motor function, improved cognitive function, improved energy (energy, reduced fatigue), improved sense of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as a result of the treatments described herein) can also be assessed by obtaining samples of cancer cells from the site of the tumor, such as a pancreatic adenocarcinoma (e.g., over the course of treatment), and testing the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells and, at a molecular level, to determine whether the cancer cells are regressing insufficiently to a malignant phenotype. For example, tumor regression induced using the methods of the present invention is indicated by a decrease in any of the pro-angiogenic markers discussed above, an increase in the anti-angiogenic markers described herein, or the discovery of a normalization (i.e., a change to the state found in normal individuals without cancer) of a metabolic pathway, an intercellular signaling pathway, or an intracellular signaling pathway that exhibits abnormal activity in an individual diagnosed with cancer. One of skill in the art will appreciate that in some embodiments, a therapeutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0153] Mutants:As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that exhibits substantial structural identity with a reference molecule, but is structurally distinct from the reference molecule, e.g., in the presence or absence, or level, of one or more chemical moieties compared to the reference entity. In some embodiments, a variant is also functionally distinct from its reference molecule. In general, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more such characteristic structural elements, but differs in at least one aspect from the reference molecule. To give some examples, a polypeptide may have / have a designated position relative to each other in linear or three-dimensional space, or have a characteristic sequence element made up of multiple amino acids that contribute to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element made up of multiple nucleotide residues that have a designated position relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence. In some embodiments, a variant polypeptide or nucleic acid exhibits a total sequence identity with a reference polypeptide or nucleic acid that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid.

[0154] vector:As used herein, and further in the disclosure above, vector refers to a nucleic acid molecule capable of transporting linked or other nucleic acids. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain vectors are also capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above may generally be performed by conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual 2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose.

[0155] In the context of this specification, all publications, patent applications, patents and other references mentioned in this application shall be expressly incorporated herein by reference in their entirety for all purposes as if fully set forth, unless otherwise indicated, and shall be considered as part of this disclosure in their entirety.

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control.

[0157] The foregoing written description of the present invention provides ways and processes for making and using the same to enable one skilled in the art to make and use the same, and such enabling is provided with particularity to the subject matter of the appended claims, which form a part of the inherent description.

[0158] As used herein, the phrases "selected from the group consisting of," "selected from," and the like, are inclusive of mixtures of particular substances.

[0159] Where a numerical limit or range is stated herein, the endpoints are included, and all values ​​and subranges within the numerical limit or range are specifically included as if expressly written.

[0160] The above description is provided to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiment will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0161] Without being bound to the specific embodiments below, the present invention is exemplified by the following: (1) A bispecific epitope binding protein comprising a combination of (VH), (VL), (X), (VH)n, (VL)n, (X)n, (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m; wherein VH and VL refer to the variable heavy chain region and the variable light chain region, respectively, of an antibody or antigen-binding fragment thereof that binds to 4-1BB; X is a PD-1 protein or a fragment thereof that binds to PD-L1; Y represents a fragment of a heavy chain constant region or a light chain constant region comprising one or more domains selected from the group consisting of CL, CH1, CH2, and CH3; n and m are each an integer of 1 to 5.

[0162] (2) In (1), (VH), (VL), (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m, if present, may be presented in any order from N-terminus to C-terminus.

[0163] (3) In (1) or (2), when multiple copies of (VH), (VL), (VH)nY, (VL)nY, (VH-VL)nY, (VL-VH)nY, (X)nY, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m are present, they can be in any order relative to the other regions and need not be directly adjacent to regions of a similar type.

[0164] (4) The bispecific epitope binding protein of any one of (1) to (3), wherein the bispecific epitope binding protein is in scFv form.

[0165] (5) The bispecific epitope binding protein of any one of (1) to (4), wherein the bispecific epitope binding protein has the structure An-A'n'-B-Cm-C'm', where: A, A', C, C': VH, VL, VH-VL, VL-VH or X B:Y VH, VL, X, Y, n, and m are as defined above, and each VH, VL, X, and Y is linked directly or via a linker and / or hinge; A dual specific epitope binding protein, wherein n' and m' are independently integers from 1 to 5.

[0166] (6) Any one of (1) to (4) above, wherein the dual specific epitope binding protein has the structure An-B-Cm, where: A or C: VH-VL, VL-VH or X B:Y A dual specific epitope binding protein, wherein VH, VL, X, Y, n, and m are as defined above, and each VH, VL, X, Y is linked directly or by a linker and / or hinge.

[0167] (7) A dual specific epitope binding protein according to any one of (1) to (4), wherein the CH1 domain is linked at the C-terminus to (VH-VL)n, (VL-VH)n, or (X)n directly or via a hinge and / or linker, and the CH2 / CH3 domain is linked at the N-terminus to (VH-VL)m, (VL-VH)m, (X)m directly or via a hinge and / or linker, wherein the VH, VL, X, n, and m are as defined above.

[0168] (8) A dual specific epitope binding protein according to (7), wherein the CH1 domain is linked to the CH2 / CH3 domains either directly or by a hinge and / or linker.

[0169] (9) The dual specific epitope binding protein of any one of (1) to (3), wherein the dual specific epitope binding protein comprises: -CH2 comprises VH-VL, VL-VH, X, and / or VH-CH1 linked directly or through a linker and / or hinge, and independently, CH3 comprises a heavy chain and a light chain comprising a VL linked to a CL via a linker and / or hinge wherein said VH, VL, and X are as defined above, and each VH, VL, X, CH1, CH2, CH3, and CL are linked directly or via a linker and / or hinge.

[0170] (10) A dual specific epitope binding protein comprising (VH and VL, in any order) nY and / or Y-(X)m in any one of (1) to (4), wherein said Y is independently a CH2 or CH3 domain, and VH, and VL and CH2 and / or CH3, or CH2 and / or CH3 and X, are linked directly or through a hinge and / or linker; VH, VL, X, n, and m are dual specific epitope binding proteins as defined above.

[0171] (11) The bispecific epitope binding protein of any one of (1) to (4) and (10), wherein the bispecific epitope binding protein is an scFv arranged from N-terminus to C-terminus as follows: (VL-VH)n-CH2-CH3-(X)m.

[0172] (12) The dual specific epitope binding protein of (11), wherein VL and VH, VH and CH2, CH3 and (X)m, and / or CH2 and CH3 are linked directly or by a linker and / or hinge.

[0173] (13) A dual specific epitope binding protein comprising (X)mY and / or Y-(VH and VL, in any order)n according to any one of (1) to (4), wherein said Y, is independently a CH2 or CH3 Domain, and wherein VH, and VL and CH2 and / or CH3, or CH2 and / or CH3 and X, are linked directly or through a hinge and / or linker; A dual specific epitope binding protein, wherein VH, VL, X, n, and m are as defined above.

[0174] (14) The bispecific epitope binding protein of (13), wherein the bispecific epitope binding protein is an scFv arranged from N-terminus to C-terminus as follows: (X)m-CH2-CH3-(VH-VL)n.

[0175] (15) The bispecific epitope-binding protein of (14), wherein (X)m and CH2, CH3 and VH, VH and VL, and / or CH2 and CH3 are linked directly or by a linker and / or hinge.

[0176] (16) The dual specific epitope binding protein of any one of (1) to (15), wherein each n and m is independently selected from the integers 1, 2, 3, 4, or 5, and any combination of n and m having such integer values ​​can be present.

[0177] (17) A bispecific epitope binding protein according to any one of (1) to (16), wherein when multiple VH, VL, and / or X are present (i.e., when n and / or m are greater than 1), the copies are directly linked or separated by hinge and / or linker sequences.

[0178] (18) A dual specific epitope binding protein in (17), wherein the copies are separated by a linker sequence.

[0179] (19) The dual specific epitope binding protein of any one of (5) to (18), wherein the linker comprises about 10 to 25 amino acid residues, 10 to 20 amino acid residues, or 12 to 19 amino acid residues.

[0180] (20) In any one of (5) to (19), the linker is (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), a 218 linker, or a 218S linker.

[0181] (21) A dual specific epitope binding protein according to any one of (5) to (20), wherein the linker is a (G4S)3 linker, a (G4S)2 linker, a 218 linker, or a 218S linker.

[0182] (22) The bispecific epitope binding protein of any one of (1) to (21), wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:23.

[0183] (23) A bispecific epitope binding protein according to any one of (1) to (22), wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO:25.

[0184] (24) A bispecific epitope binding protein according to any one of (1) to (21), wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:23 and a VL of SEQ ID NO:25.

[0185] (25) The dual specific epitope binding protein of any one of (1) to (24), wherein the PD-1 protein or fragment thereof comprises the sequence of SEQ ID NO:27.

[0186] (26) The bispecific epitope binding protein of any one of (1) to (21), wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:23 and a VL of SEQ ID NO:25, and the PD-1 protein or fragment thereof comprises a sequence of SEQ ID NO:27.

[0187] (27) The bispecific epitope binding protein of any one of (1) to (26), wherein the bispecific epitope binding protein has a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14, or the bispecific epitope binding protein has at least 80% sequence identity to a bispecific epitope binding protein having a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14, and has bispecific binding affinity for 4-1BB and PD-L1.

[0188] (28) The dual specific epitope binding protein of any one of (1) to (26), wherein the dual specific epitope binding protein has a sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and 14.

[0189] (29) The bispecific epitope binding protein of any one of (1) to (26), wherein the bispecific epitope binding protein has at least 80% sequence identity to a bispecific epitope binding protein having a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14, and has bispecific binding affinity for 4-1BB and PD-L1.

[0190] (30) The dual specific epitope binding protein of any one of (1) to (26), wherein the dual specific epitope binding protein has the sequence of SEQ ID NO:3.

[0191] (31) The bispecific epitope binding protein of any one of (1) to (26), wherein the bispecific epitope binding protein has at least 80% sequence identity to a bispecific epitope binding protein having a sequence of SEQ ID NO:3 and has bispecific binding affinity for 4-1BB and PD-L1.

[0192] (32) The dual specific epitope binding protein according to any one of (1) to (31), further comprising a signal peptide.

[0193] (33) The dual specific epitope binding protein of (32), wherein the signal peptide is an N-terminal signal peptide comprising 12 to 40 amino acids.

[0194] (34) A dual specific epitope binding protein according to any one of (5) to (33), wherein the hinge has the sequence of SEQ ID NO:30 or SEQ ID NO:31.

[0195] (35) A dual specific epitope binding protein according to any one of (5) to (34), wherein the hinge has the sequence of SEQ ID NO:31.

[0196] (36) A bispecific epitope binding protein comprising any one of (1) to (35) above, wherein the PD-1 protein or a fragment thereof is affinity matured.

[0197] (37) A dual-specific epitope-binding protein pair according to any one of (1) to (36), wherein both members of the pair are identical.

[0198] (38) A nucleic acid molecule encoding a dual specific epitope binding protein according to any one of (1) to (36).

[0199] (39) A recombinant vector comprising the nucleic acid molecule of (38).

[0200] (40) A cell comprising the nucleic acid molecule of (38) or the recombinant vector of (28).

[0201] (41) A method for producing a bispecific epitope binding protein, the method comprising culturing a cell of (40) in a culture medium under conditions sufficient to produce the bispecific epitope binding protein; and recovering the bispecific epitope binding protein from the cells and / or the culture medium.

[0202] (42) A pharmaceutical composition comprising a bispecific epitope binding protein according to any one of (1) to (36), a nucleic acid according to (38), a recombinant vector according to (39), or a cell according to (40), and a pharma- ceutical acceptable carrier.

[0203] (43) A kit comprising the pharmaceutical composition according to (42).

[0204] (44) A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising or transmitting an effective amount of a bispecific epitope binding protein of (1)-(36), a nucleic acid of (37), a recombinant vector of (38), or a cell of (40).

[0205] (45) The method according to (44), wherein the subject has cancer.

[0206] (46) The method according to (44), wherein the subject is at risk of developing cancer.

[0207] (47) The method according to any one of (44) to (46), wherein the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, pharyngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, prostate cancer, and combinations thereof.

[0208] (48) A method for preventing cancer in a subject in need thereof, the method comprising the step of administering to the subject a composition comprising or transmitting an effective amount of a bispecific epitope binding protein of (1)-(36), a nucleic acid of (38), a recombinant vector of (39), or a cell of (40).

[0209] (49) The method according to (48), wherein the subject has cancer.

[0210] (50) The method according to (48), wherein the subject is at risk of developing cancer.

[0211] (51) The method according to any one of (48) to (50), wherein the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, pharyngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, prostate cancer, and combinations thereof.

[0212] (52) A method according to any one of (48) to (51), wherein the prevention suppresses the onset of clinical symptoms of cancer.

[0213] (53) The method according to any one of (48) to (51), wherein the prevention is the alleviation of clinical symptoms associated with cancer.

[0214] (54) The method according to any one of (47) to (50), wherein the prevention is the elimination of clinical symptoms associated with cancer.

[0215] Having generally described the present invention, an additional understanding can be obtained with reference to certain specific examples, which are provided herein for illustrative purposes only and are not intended to be limiting, unless otherwise expressly stated. EXAMPLES

[0216] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0217] Example 1: Bispecific antibody (BsAB) design and production For the anti-4-1BB antibody, the 94kvt clone was used (EU101 described in International Publication No. WO2018-127787, the entire contents of which are incorporated herein by reference), and for the anti-PD-L1 protein, an affinity matured PD-1 protein (euPD-1, EU131 described in U.S. Patent No. 17 / 469,549, the entire contents of which are incorporated herein by reference) was used. A total of 10 (S01-S10) anti-4-1BB antibody x euPD-1 mutants (referred to herein as "dual-specific epitope binding protein mutants") were designed (SEQ ID NOs: 1-10, 14; dual-specific epitope binding protein S01-S10 and 94kvt light chain, respectively) with altered sequences, using (G4S)3, (G4S)2, and 218 or 218S (SEQ ID NOs: 15-18) as linkers to link each moiety. In the case of 94kvt, an scFv format was used. An engineered IgG1 hinge and Fc portion was used to link with euPD-1. In the case of Fc, point mutations (L234A, L235A, K322A, D356E, L358M) were introduced to reduce ADCC and CDC. (Figure 2)

[0218] In addition, the S11 (SEQ ID NO:11) antibody was developed to be used as a control by attaching a commercially available anti-PD-L1 antibody (atezolizumab, Genentech) in the form of an scFv to the C-terminus of the antibody (Figure 2).

[0219] Expression of the generated bispecific antibodies was accomplished using the Expi293F Expression System (Invitrogen) or ExpiCHO Expression System (ThermoFisher scientific). Purification was accomplished using an AktaPure (cytiva) instrument, a protein A column (Hitrap MabSelectSure, cytiva, Cat.no.11003495) and a desalting column (Hiprep 26 / 10 Desalting, Cytiva Cat.no.17508702).

[0220] Example 2: SDS-PAGE analysis of dual specific epitope binding protein variants SDS-PAGE analysis was performed to analyze the dual specific epitope binding protein variants under reducing and non-reducing conditions. Reducing conditions consisted of adding 1 μg of the dual specific epitope binding protein variants to LDS sample buffer (Invitrogen, Cat. No. NP0007) with sample reducing agent (Thermo, Cat. No. NP0009), followed by boiling at 95°C for 3 minutes. For non-reducing conditions, only LDS sample buffer was added. Samples generated on TGX stain-free gels (Mini-PROTEAN TGX stain-free gels) were loaded onto the gel and run in 1x Tris / glycine / SDS buffer (Bio-Rad, Cat. No. 1610732) at 125V for 30 minutes. The results were analyzed using Chemidoc (Bio-Rad) and are shown in Figure 3.

[0221] Example 3: Size-exclusion chromatography analysis of dual specific epitope binding protein variants The dual specific epitope binding protein variants were also applied to SEC analysis using an HPLC instrument (Agilent Technologies, 1260InfinityII LC system) and a size exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8×300 mm, Part No. 0008541, Column No. 004E04320E). A gel filtration standard (Bio-rad, Cat no. 1511901) was used as a control. The results are illustrated in FIG. 4.

[0222] Example 4: Affinity analysis of dual specific epitope binding protein variants Surface plasmon resonance (SPR) was used to analyze the affinity of the dual-specific epitope binding protein variants.

[0223] Affinity for 4-1BB For 4-1BB affinity determination, the bispecific epitope binding protein variants diluted at 5 μg / ml were immobilized on a CM5 chip (Cytiva, BR-1005-30) using human capture (Cytiva, Cat. No. BR-1008-39). 4-1BB antigen (abclon) was injected at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 nM, and the binding time was set to 150 seconds and the dissociation time was set to 240 seconds. Biacore T200 (cytiva) was used as the analytical equipment. Table 1 shows the results of 4-1BB affinity evaluation: [Table 1]

[0224] Affinity for PD-L1 For PD-L1 affinity determination, bispecific epitope-binding protein variants diluted at 10 μg / ml were immobilized on a CM5 chip using human capture. PD-L1 antigen (SinoBiologics, Cat. No. 10084-H08H) was injected at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 nM, and the subsequent procedures were identical to the 4-1BB affinity analysis procedures. Table 2 shows the results of PD-L1 affinity evaluation: [Table 2]

[0225] To compare the binding difference between native PD-1 and S03 to PD-L1, and the binding of mouse PD-1 and human PD-L1, human PD-1-Fc (SinoBiologics) and mouse PD-1 Fc (SinoBiologics, Cat No. 50124-M02H) were diluted to 15 μg / ml and immobilized on a CM5 chip with human capture. PD-L1 antigen was injected at concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM, and the subsequent procedures were identical to the 4-1BB affinity analysis procedures. The results are shown in Figure 5a for human native PD1-Fc and in Figure 5b for S03. S03 was confirmed to bind PD-L1 approximately 93 times more strongly than native PD-1. The affinity of mouse PD-1 to human PD-L1 was not confirmed (see Table 3).

[0226] [Table 3]

[0227] Example 5: Single antigen binding ELISA To confirm whether the dual specific epitope binding protein variants bind to each target antigen, single antigen binding ELISA was performed. The assay method was performed as shown in Figure 6a.

[0228] To confirm 4-1BB binding, a 96-well immunoplate was coated with 1 μg / ml 4-1BB antigen (abclon) overnight at 4° C., and 150 μl of 1× assay buffer (BioLegend) was added for 1 hour to block non-specific binding. The bispecific epitope binding protein variant was diluted 4-fold at 3 nM for 4 places, and 100 μl per well was added for 2 hours. Next, HRP-conjugated anti-human IgG, Fcγ antibody (Jackson Immunoresearch, Cat. No. 10-035-008) was diluted 2000-fold and added 100 μl per well and incubated for 1 hour. After color development with TMB, the reaction was stopped using sulfuric acid. All steps except the TMB treatment step were washed three times with washing buffer, and all steps except the coating step were carried out at room temperature. (Figure 6b)

[0229] To confirm PD-L1 binding, 96-well immunoplates were coated with 1 μg / ml PD-L1 antigen (SinoBiologics) overnight at 4℃, and 150 μl of 1x assay buffer (BioLegend) was added for 1 hour to block non-specific binding. The bispecific epitope binding protein variants were diluted 10-fold in quadruplicates at 300 nM, and then 100 μl per well was treated for 2 hours. The rest of the procedure was the same as that of 4-1BB binding ELISA (Fig. 6c).

[0230] In addition, to confirm the specific binding to each antigen-binding site, i) the anti-4-1BB antibody of S03 was replaced with human germline antibody (DP47) to prevent binding to 4-1BB, to prepare S12 (SEQ ID NO: 12), and ii) the euPD-1 of S03 was point mutated and replaced with euPD-1n to prepare S13 (SEQ ID NO: 13) to prevent binding to PD-L1, and single antigen-binding ELISA for 4-1BB and PD-L1 was performed (Figure 6d).

[0231] For 4-1BB binding, S03, S12, and S13 were diluted 2-fold at 0.5 μg / ml and examined at a total of 11 sites. S12, which does not have anti-4-1BB antibodies, was unable to bind to 4-1BB, while S03 and S13 were confirmed to bind to 4-1BB in a dose-dependent manner (Figure 6e).

[0232] For PD-L1 binding, S03, S12 and S13 were diluted 4-fold at 100μg / ml to examine 11 sites in total. We noted that S13, which has a point mutation that renders it unable to bind to PD-L1, did not bind to PD-L1, whereas S03 and S12 bound to PD-L1 in a dose-dependent manner (Fig. 6f).

[0233] Example 6: Double antigen-binding ELISA To confirm that the dual specific epitope binding protein variants bind to both antigens 4-1BB and PD-L1, dual binding assays were performed.

[0234] The assay method was performed as shown in Figure 7a. Briefly, 96-well immunoplates were coated with 1μg / ml PD-L1 antigen (SinoBiologics) overnight at 4℃, and 150μl of 1x assay buffer (BioLegend) was added for 1 hour to block non-specific binding. Bispecific epitope binding protein variants were diluted 5-fold from 10nM in 4 increments, 100μl per well was added for 2 hours, and 100μl of biotinylated 4-1BB antigen (SinoBiologics, Cat. No. 10041-H08H-B) at 0.1μg / ml was added to each well and incubated for 1 hour. Next, avidin-HRP (BioLegend, Cat No. 79004) was diluted 1 / 1000 and added to each well at 100 μl for 30 minutes. After color development with TMB, the reaction was stopped using sulfuric acid. After the TMB addition, all steps were washed three times with washing buffer, and all steps except the coating step were carried out at room temperature (Figure 7b).

[0235] A double antigen ELISA for S03, S12, and S13 was also performed at 100μg / ml, diluted 12 times by 5, to confirm specific binding by each antigen-binding moiety. S12, which has no anti-4-1BB antibody, and S13, which has a point mutation that prevents it from binding to PD-L1, did not show double antigen binding, indicating that S03 binds in a dose-dependent manner (Figure 7c).

[0236] Example 7: Cell Binding Assay PD-L1 positive cell lines MDA-MB-231-Luc (human breast cancer cells, JCRB, JCRB1559), FaDu (human squamous cell carcinoma cells, ATCC, HTB-43), 4-1BB positive cell line 4-1BB Jurkat (self-produced), and PD-L1 and 4-1BB negative cell line MCF-7 (human breast cancer cells) were used for analysis. Figure 8 shows the expression levels of 4-1BB and PD-L1 in each cell line, which were determined by the secondary antibody with little background signal.

[0237] 2 × 10 cells from each cell line 5 Individual cells were treated with the bispecific epitope binding protein variants at final concentrations of 50, 5, 0.5, and 0.05 nM, followed by incubation at 4° C. for 20 minutes. Then, one wash was performed using FACS wash buffer, and 1 μl / tube of anti-hFC-488 secondary antibody (Jackson ImmunoResearch) was treated for 20 minutes. After two washes, analysis was performed using FACS (FACSCelesta, BD).

[0238] FACS analysis revealed that the dual-specific epitope binding protein variants bound to PD-L1 (Figures 9a-9b) and 4-1BB positive cell lines (Figure 9c) in a dose-dependent manner, but did not bind to cells that neither expressed 4-1BB nor PD-L1 (Figure 9d).

[0239] Example 8: 4-1BB Bioanalysis To verify the activity of 4-1BB, a 4-1BB bioassay (Promega, J2332) was performed based on the principle that luciferase is expressed when 4-1BB effector cells are activated.

[0240] Bioassays and luciferase assays were performed according to the manufacturer's protocols. MDA-MB-231 cells expressing PD-L1 were dispensed into 96-well white plates (Costar). After overnight incubation in a 37°C CO2 incubator, the bispecific epitope binding protein variants and 4-1BB effector cells were treated. The antibodies were diluted 2-fold starting from 100ng / ml and run in quadruplicate. After treatment with 4-1BB effector cells, the cells were incubated for 6 hours in a 37°C CO2 incubator and the luminescence intensity was measured using a luminometer (Floroskan Ascent FL, Thermo). The results are shown in Figure 10.

[0241] Example 9: 4-1BB / PD-1 Combination Bioanalysis Based on the principle that luciferase is expressed when effector cell binding to PD-1 and PD-L1 is blocked and 4-1BB is activated, a 4-1BB / PD-1 combination bioassay (Promega, CS1978I10) was performed to verify the inhibitory effect of PD-1 and PD-L1 binding and the effect of 4-1BB activity (Figure 11a).

[0242] Bioassays and luciferase assays were performed according to the manufacturer's protocols. PD-L1 aAPC / CHO-K1 cells expressing PD-L1 were dispensed into a 96-well white plate. They were cultured overnight in a 37°C CO2 incubator to treat the antibodies and effector cells. S03, anti-4-1BB antibody (94kvt-engineered IgG1, SEQ ID NO:14 and 21), anti-PD-1 antibody (pembrolizumab, MSD), anti-PD-L1 antibody (atezolizumab, Roche), and euPD-1 Fc (SEQ ID NO:22) were used. Seven 3-fold dilutions were performed at 147nM. 4-1BB effector cells were added for treatment and incubated for 6 hours in a 37°C CO2 incubator, and the luminescence intensity was measured using a luminometer (Floroskan Ascent FL, Thermo).

[0243] The experimental results (Figure 11b) confirmed that strong luminescence was detected by S03, which can bind to PD-L1-expressing cells and effector cells.

[0244] Example 10: Affinity for PD-L1 or PD-L2 PD-1 is known to bind to both PD-L1 and PD-L2. S03 contains euPD-1, an affinity-matured form of PD-1. The binding of S03 to PD-L1 and PD-L2 was confirmed by ELISA.

[0245] 96-well immunoplates were coated with 2μg / ml human PD-1-Fc (SinoBiologics, Cat. No. 10377-H02H) at 100μl per well overnight at 4℃ and treated with 150μl of 1x assay buffer (BioLegend) for 1 hour to block non-specific binding. Biotinylated PD-L1 (SinoBiologics, Cat. No. 10084-H08H-B) and biotinylated PD-L2 (SinoBiologics, Cat. No. 10292-H08H-B) were diluted 3-fold from 1μg / ml in five increments at 100μl per well for 1 hour. Avidin-HRP (BioLegend, Cat No. 79004) was diluted 1 / 1000 and added at 100 ul per well for 30 minutes, and then the plate was colored with TMB and the reaction was stopped with sulfuric acid. All steps except the step after TMB treatment were washed three times with washing buffer, and all steps except the antibody coating step were carried out at room temperature.

[0246] In addition, S03, anti-PD-L1 antibody (Roche), and anti-PD-L2 antibody (SinoBiologics, Cat. No. 10292-R018) were diluted 4 times at 0.1μg / ml, and 100μl was added per well to a 96-well immunoplate and coated overnight at 4℃. Then, 150μl of 1x assay buffer was added for 1 hour to block non-specific binding. 100μl of biotinylated PD-L1 and biotinylated PD-L2 were added to each well at a concentration of 0.1μl / ml for 1 hour. The rest of the procedure was the same as human PD-1-Fc ELISA.

[0247] The experimental results showed that human PD-1-Fc bound to both PD-L1 and PD-L2 (Fig. 12a), whereas the control anti-PD-L1 antibody was shown to bind only to PD-L1, and the control anti-PD-L2 antibody was shown to bind only to PD-L2. Test substance S03 only bound to PD-L1 (Fig. 12b-c).

[0248] Example 11: Cytotoxicity Assay To confirm the T cell-mediated cytotoxic effect of S03 on target cells, a cytotoxicity assay was performed. T cells isolated from human peripheral blood mononuclear cells by pan T negative selection (Miltenyi Biotec, Cat. No. 130-096-535) were activated on plates coated with anti-CD3 antibody (OKT3, Bio X Cell) for 4 days. One day before T cell and antibody treatment, the PD-L1 positive cell line MDA-MB-231-GFP (self-produced) was added at 4 × 10 4 Anti-4-1BB antibody (94kvt-engineered IgG1), anti-PD-1 antibody (pembrolizumab, MSD) and S03 were diluted 3-fold at 1.667 nM in four doses per well. Activated T cells were harvested and then treated with tumor cells in a 1:1 ratio. GFP signals were measured at hourly intervals for 117 hours using a live cell analysis system (Incucyte, Sartorius).

[0249] The experimental results showed that the data was for 1.667 nM, confirming that S03 had a superior T cell-mediated cytotoxic effect compared to the anti-4-1BB antibody and anti-PD-L1 antibody used as controls (Figure 13).

[0250] Example 12: IFN-γ Analysis The level of IFN-γ produced by T cells was measured using the supernatant obtained from the cytotoxicity assay (Example 11). An IFN-γ ELISA kit (BioLegend, Cat. No. 430101) was used according to the manufacturer's protocol. In brief, 150 μl of 1x assay buffer (BioLegend) was applied to the plate coated overnight with the capture antibody for 1 hour. The standard was used after diluting 7 times from 500 pg / ml, and the supernatant was diluted 50 times and 100 μl was applied to each well for 2 hours. After applying 100 μl of detection antibody for 1 hour, avidin-HRP (BioLegend, Cat. No. 79004) was diluted 1 / 1000 and applied to each well for 30 minutes. After color development with TMB, the reaction was stopped using sulfuric acid. All steps except the post-TMB treatment step were washed three times with wash buffer, and all steps except the antibody coating step were carried out at room temperature.

[0251] As a result of the experiment, it can be observed that IFN-γ production by T cells increases depending on the concentration of antibody treatment (Figure 14).

[0252] Example 13: Antibody-dependent cell cytotoxicity assay The IgG1-Fc engineering of the bispecific epitope binding protein mutants was used to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). To confirm whether ADCC was reduced, an ADCC bioassay (Promega, Cat. No. G701A) was performed using S03.

[0253] To confirm ADCC due to the binding of anti-4-1BB antibody S03, 4-1BB positive cell line (4-1BB NF-kB HEK293) was dispensed into a white 96-well plate (Costar) and cultured overnight. The next day, anti-4-1BB antibodies S03 (engineered IgG1, L234A, L235A, K322A, D356E, L358M), 94kvt-IgG1 (SEQ ID NO: 14 and 20), and 94kvt-engineered IgG1 (L234A, L235A, K322A) were diluted 3-fold from 294 nM to 8 dilutions. ADCC effector cells were added and incubated for 6 hours in a 37°C incubator. Bio-Glo luciferase substrate was then added and the luminescence intensity was measured using a luminometer (Glo-max, Promega). It can be observed that 94kvt-IgG1, used as a positive control, induces ADCC, whereas the negative controls 94kvt-engineered IgG1 and S03 do not induce ADCC (Figure 15a).

[0254] To confirm the ADCC of S03 by euPD-1, PD-L1-positive cell lines (MDA-MB-231) were plated in white 96-well plates (Costar) and cultured overnight. The next day, anti-PD-L1 antibodies S03, Avelumab (Pfizer, IgG1), and Atezolizumab (Roche, engineered IgG1, N297A) were diluted 4-fold in six doses starting from 5.88 nM. ADCC effector cells were added and incubated for 6 hours in a 37°C incubator. Bio-Glo luciferase substrate was then added and the luminescence intensity was measured using a luminometer (Glo-max, Promega). It can be seen that Avelumab, used as a positive control, induces ADCC, whereas Atezolizumab and S03, which are negative controls, do not induce ADCC (Figure 15b).

[0255] Example 14: Complement-dependent cytotoxicity assay C1q ELISA was performed to confirm that CDC was reduced by the engineered IgG1 used in S03.

[0256] A 96-well immunoplate (NUNC) was coated with 75 nM S03, Avelumab, and Atezolizumab overnight at 4°C using 1x coating buffer (BioLegend), and then 150 μl of 1x analysis buffer (BioLegend) was added for 1 hour to block non-specific binding. Biotinylation kit (Abcam, Cat. No. Ab201795) was used to dilute biotinylated C1q (comp Tech, Cat. No. A099) protein from 2 μg / ml in 7 2-fold increments, and 100 μl was added per well for 2 hours. Next, avidin-HRP (BioLegend, Cat. No. 79004) was diluted 1 / 1000 and 100 μl was added to each well for 1 hour, after which the plate was developed with TMB and the reaction was stopped using sulfuric acid. All steps except the TMB post-treatment step were washed three times using washing buffer, and all steps except the antibody coating step were carried out at room temperature. It can be observed that Avelumab, used as a positive control, binds to C1q, while S03 and Atezolizumab, the negative control, do not bind to C1q (Figure 16).

[0257] Example 15: In vivo efficacy study 1-4-1BB KI To verify the efficacy of S03, in vivo studies were performed using h4-1BB knock-in mice (background: C57BL / 6, Biocytogen).

[0258] MC38 cells expressing PD-L1 were used as tumor cells as shown in Figure 17a, and 5 × 10 5 Tumor cells were administered subcutaneously. One week after administration, the size of the tumors was measured and the mean size (100 mm) was similar. 3 Treatment groups were divided based on the mean mean (within 100%) and standard deviation. Five mice were used for each group.

[0259] S03, S05 and S07 were injected intravenously. Vehicle DPBS (Gibco) was used as a negative control. Antibody doses were set at 2mg / kg and 5mg / kg. Administration was performed in 100μl, and five doses were administered at three-day intervals. Tumor size was also measured at three-day intervals, and tumor size was measured up to two days after the last administration.

[0260] As a result of tumor size observation, as shown in Figure 17b, it was confirmed that S03, S05, and S07 all suppressed tumor growth compared to the negative control group, and among them, S03 had the most excellent tumor reduction effect. In addition, a dose-dependent response was observed for the tested antibodies.

[0261] Example 16: In vivo efficacy study 2-4-1BB KI To verify the efficacy of S03, an in vivo study was carried out using h4-1BB knock-in mice (background: C57BL / 6, Biocytogen). The test method was the same as in Figure 17a.

[0262] S03, anti-4-1BB antibody (94kvt-engineered IgG1), and anti-PD-L1 antibody (atezolizumab) were intravenously injected. Vehicle DPBS (Gibco) was used as a negative control. The antibody dose was set at 5mg / kg (5.88μM) based on S03, and 5.88μM of anti-4-1BB antibody and anti-PD-L1 antibody were also administered. Administration was performed in 100μl, and five doses were administered at three-day intervals. Tumor size was also measured at three-day intervals, and tumor size was measured up to two days after the last administration. Blood was also collected on the day of the end of the experiment, and the concentrations of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and BUN (serum urea nitrogen) in the blood were confirmed using a biochemical analyzer to confirm toxicity.

[0263] As a result of observing the tumor size, as shown in Figure 18a, it was confirmed that S03, anti-4-1BB antibody, and anti-PD-L1 antibody all suppressed tumor growth compared to the negative control group, and among them, S03 had the most excellent tumor reduction effect (approximately 98% reduction).

[0264] As shown in FIG. 18b, the blood T cell population was analyzed, and blood CD4+ T cells and CD8 + T cells were increased by S03, and an increase in effector T cells was observed among CD8+ T cells.

[0265] As shown in Table 4, the results of the hepatotoxicity index analysis showed that all four indices in the treatment group were within the normal range (ALT: 17-77 U / L, AST: 54-298 U / L, BUN: 8-33 mg / dL), confirming that there was no hepatotoxicity in the liver under the experimental conditions. [Table 4]

[0266] Example 17: In vivo efficacy study 3-h4-1BB / hPD-1 DKI To verify the efficacy of S03, in vivo studies were carried out using h4-1BB and hPD-1 double knock-in mice (background: C57BL / 6, Biocytogen).

[0267] MC38 cells expressing PD-L1 were used as tumor cells as shown in Fig. 17a, and 5 × 10 5 Tumor cells were administered subcutaneously. One week after administration, the size of the tumors was measured and they had a similar average size (approximately 100 mm 3 The treatment groups were divided based on the mean mean and standard deviation. Six mice were used in each group.

[0268] Antibody S03, anti-4-1BB antibody (94kvt-engineered IgG1), and anti-PD-L1 antibody (atezolizumab) were injected intravenously. Vehicle DPBS (Gibco) was used as a negative control group. Antibody dose was set at 5mg / kg (5.88μM) based on S03, and 5.88μM of anti-4-1BB antibody and anti-PD-L1 antibody were also administered. Administration was performed in 100μl, and five doses were administered at 3-day intervals. Tumor size was also measured at 3-day intervals, and tumor size was measured up to 2 days after the last administration.

[0269] As a result of observing the tumor size, as shown in Figure 19, it was confirmed that tumor growth was suppressed by all of S03, anti-4-1BB antibody, and anti-PD-L1 antibody compared to the negative control group (a reduction of approximately 73%).

[0270] All data are presented graphically with mean SEM, one-way ANOVA statistical test. All statistics were calculated using the mean data per animal or sample. TIFF2024502371000006.tif9170

[0271] The above describes the specific parts of the present invention. It is obvious that such specific description is merely a preferred embodiment for those skilled in the art, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention is intended to be defined by the appended claims and their equivalents.

Claims

1. 13. A dual specific epitope binding protein comprising a combination of: (VH), (VL), (X), (VH)n, (VL)n, (X)n, (VH)n-Y, (VL)n-Y, (VH-VL)n-Y, (VL-VH)n-Y, (X)n-Y, Y-(VH)m, Y-(VL)m, Y-(VH-VL)m, Y-(VL-VH)m, and / or Y-(X)m, VH and VL refer to the variable heavy and light chain regions, respectively, of an antibody or antigen-binding fragment thereof that binds to 4-1BB; X is a PD-1 protein or a fragment thereof that binds to PD-L1; Y represents a fragment of a heavy chain constant region or a light chain constant region comprising one or more domains selected from the group consisting of CL, CH1, CH2, and CH3; A dual specific epitope binding protein, wherein n and m are each integers from 1 to 5.

2. 2. The dual specific epitope binding protein of claim 1, wherein (VH), (VL), (VH)n-Y, (VL)n-Y, (VH-VL)n-Y, (VL-VH)n-Y, (X)n-Y, Y-(VH), Y-(VL), Y-(VH-VL), Y-(VL-VH), and / or Y-(X)m, when present, may be presented in any order from N-terminus to C-terminus.

3. A dual specific epitope binding protein having the structure An-A'n'-B-Cm-C'm', Where: A, A', C, C': VH, VL, VH-VL, VL-VH or X; B: Y; VH, VL, X, Y, n, and m are as defined above, and each VH, VL, X, and Y is linked directly or via a linker and / or hinge; 2. The dual specific epitope binding protein of claim 1, wherein n' and m' are independently integers from 1 to 5.

4. 1. A dual specific epitope binding protein having the structure An-B-Cm, Where: A or C: VH-VL, VL-VH or X; B: Y; 2. The dual specific epitope binding protein of claim 1, wherein VH, VL, X, Y, n, and m are as defined above, and each VH, VL, X, Y is linked directly or by a linker and / or hinge.

5. 2. The dual specific epitope binding protein of claim 1, wherein the CH1 domain is linked C-terminally to (VH-VL)n, (VL-VH)n, or (X)n directly or by a hinge and / or linker, and the CH2 / CH3 domains are linked N-terminally to (VH-VL)m, (VL-VH)m, (X)m directly or by a hinge and / or linker, wherein said VH, VL, X, n, and m are as defined above.

6. The dual specific epitope binding protein comprises: -CH2 comprises a VH-VL, VL-VH, X, and / or VH-CH1 linked directly or through a linker and / or hinge to CH3, and independently, CH4 comprises a heavy chain and - comprising a light chain comprising a VL linked directly or through a linker and / or hinge to a CL, 2. The dual specific epitope binding protein of claim 1, wherein said VH, VL, and X are as defined above, and each VH, VL, X, CH1, CH2, CH3, and CL are linked directly or by a linker and / or hinge.

7. 1. A dual specific epitope binding protein comprising (VH and VL, in any order) n-Y and / or Y-(X)m, said Y, is independently a CH2 or CH3 Domain, and VH, and VL and CH2 and / or CH3, or CH2 and / or CH3 and X, are linked directly or through a hinge and / or linker; 2. The dual specific epitope binding protein of claim 1 , wherein VH, VL, X, n and m are as previously defined.

8. A dual specific epitope binding protein comprising (X)m-Y and / or Y-(VH and VL, in any order), said Y, is independently a CH2 or CH3 Domain, and VH, and VL and CH2 and / or CH3, or CH2 and / or CH3 and X, are linked directly or through a hinge and / or linker; 2. The dual specific epitope binding protein of claim 1 , wherein VH, VL, X, n and m are as previously defined.

9. 2. The dual specific epitope binding protein of claim 1, wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:

23.

10. 2. The dual specific epitope binding protein of claim 1, wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO:

25.

11. The 4-1BB-binding antibody or antigen-binding fragment thereof has the structure of SEQ ID NO:23 2. The dual specific epitope binding protein of claim 1 , comprising a VH and a VL of SEQ ID NO:

25.

12. 2. The dual specific epitope binding protein of claim 1 , wherein the PD-1 protein or fragment thereof comprises the sequence of SEQ ID NO:

27.

13. 2. The dual specific epitope binding protein of claim 1 , wherein the 4-1BB-binding antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:23 and a VL of SEQ ID NO:25, and the PD-1 protein or fragment thereof comprises the sequence of SEQ ID NO:

27.

14. 2. The bispecific epitope binding protein of claim 1, wherein the bispecific epitope binding protein has a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14, or the bispecific epitope binding protein has at least 80% sequence identity to a bispecific epitope binding protein having a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14 and has bispecific binding affinity for 4-1BB and PD-L1.

15. 2. The dual specific epitope binding protein of claim 1, wherein the dual specific epitope binding protein has a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14.

16. 2. The bispecific epitope binding protein of claim 1, wherein the bispecific epitope binding protein has at least 80% sequence identity to a bispecific epitope binding protein having a sequence selected from the group consisting of SEQ ID NOs: 1-10 and 14 and has bispecific binding affinity for 4-1BB and PD-L1.

17. 2. The dual specific epitope binding protein of claim 1, wherein the dual specific epitope binding protein has the sequence of SEQ ID NO:

3.

18. 2. The bispecific epitope binding protein of claim 1, wherein the bispecific epitope binding protein has at least 80% sequence identity to a bispecific epitope binding protein having the sequence of SEQ ID NO:3 and has bispecific binding affinity for 4-1BB and PD-L1.

19. 10. The dual specific epitope binding protein of claim 1 further comprising a signal peptide.

20. 20. The dual specific epitope binding protein of claim 19, wherein the signal peptide is an N-terminal signal peptide comprising from 12 to 40 amino acids.

21. 2. The bispecific epitope binding protein of claim 1, wherein the PD-1 protein or fragment thereof is affinity matured.

22. A nucleic acid molecule encoding the dual specific epitope binding protein of claim 1.

23. A recombinant vector comprising the nucleic acid molecule of claim 22.

24. A cell comprising the nucleic acid molecule of claim 22.

25. 1. A method for producing a dual specific epitope binding protein comprising: The method comprises culturing the cells of claim 22 in a culture medium under conditions sufficient to produce the bispecific epitope binding protein; and recovering the bispecific epitope binding protein from the cells and / or the culture medium.

26. 13. A pharmaceutical composition comprising the dual specific epitope binding protein of claim 1 and a pharma- ceutically acceptable carrier.

27. ​​The pharmaceutical composition described in claim 26, which is a pharmaceutical composition for preventing or treating cancer.

28. The pharmaceutical composition described in claim 27, wherein the cancer is a PD-L1 positive cancer.

29. 28. The pharmaceutical composition of claim 27, wherein the cancer comprises bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, pharyngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, prostate cancer, and combinations thereof.

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