Antibodies binding 4-1BB and uses thereof

Abstract

To provide an isolated monoclonal antibody that specifically binds human 4-1BB, or the antigen-binding portion thereof.SOLUTION: The present invention discloses an isolated monoclonal antibody that specifically binds human 4-1BB, or the antigen-binding portion thereof. The invention also relates to a nucleic acid molecule encoding the antibody, an expression vector, a host cell and a method for expressing the antibody. The invention further relates to a bispecific molecule, and a pharmaceutical composition comprising the antibody or the bispecific molecule, as well as a treatment method using an anti-4-1BB antibody of the disclosure.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 913,744, filed October 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present disclosure generally relates to an isolated monoclonal antibody, particularly a human monoclonal antibody, or an antigen-binding portion thereof, that functionally specifically binds to human 4-1BB. The present disclosure also provides nucleic acid molecules encoding the antibody or antigen-binding portion thereof, expression vectors, host cells, and methods for expressing the antibody or antigen-binding portion thereof. The present disclosure further provides bispecific molecules. The present disclosure also provides pharmaceutical compositions comprising the antibody or antigen-binding portion thereof, and methods of treatment using the antibody or antigen-binding portion thereof. [Background technology]

[0003] 4-1BB, also known as CD137 or TNFRSF9, is a member of the tumor necrosis factor receptor family. It is best characterized as a costimulatory molecule on the T cell surface that regulates TCR-induced T cell activation. Upon binding by its major natural ligand, 4-1BBL, expressed on activated antigen-presenting cells, 4-1BB upregulates anti-apoptotic molecules and promotes cytokine production and effector function. 4-1BB is also found on dendritic cells, activated monocytes, NK cells, neutrophils, eosinophils, and mast cells, and 4-1BB signaling has been reported to stimulate IFN-γ secretion and promote NK cell proliferation and DC activation (Cariad Chester et al.,(2018)Blood 131(1):49-57).

[0004] Meanwhile, expression of 4-1BB is observed in tumor cells and serum of cancer patients. 4-1BB expression along blood vessel walls, mainly in tumor microvasculature and at sites of inflammation within atherosclerotic areas, suggests that it may mediate leukocyte migration (Drenkard D et al., (2007) FASEB J. 21 (2): 456-463). Furthermore, ligation of 4-1BB on T regulatory cells (Tregs) can induce Treg proliferation (Zhang P et al., (2007) Scand J Immunol. 66 (4): 435-440).

[0005] To investigate the complex functions of 4-1BB, Melero et al. tested the effects of agonistic anti-4-1BB antibodies against the poorly immunogenic Ag104A sarcoma and the highly immunogenic P815 mastocytoma in 1997, and demonstrated the antitumor activity of 4-1BB agonists (Melero I et al., (1997) Nature Med. 3(6):682-685). - / - Approximately 60% of the mice developed B cell lymphoma, 4-1BBL - / - It has been reported that NK cell numbers and activity in mice were reduced (Middendorp S et al., (2009) Blood 114(11):2280-2289; Vinay D Set al., (2004) J. Immunol. 173(6):4218-4229). Injection of B16.F10 melanoma cells was associated with reduced NK cell numbers and activity in mice (Middendorp S et al., (2009) Blood 114(11):2280-2289; Vinay D Set al., (2004) J. Immunol. 173(6):4218-4229). - / - 4-1BB caused more deaths in mice + / +In mice, it did not cause any adverse events (Ju SA et al.,(2005)Immunol.Cell Biol.83(4):344-351). These findings have led to 4-1BB emerging as a potent immune system stimulant and potential target for immunotherapy. Studies have shown that 4-1BB agonists can be used to treat tumors, viral infections, autoimmune diseases, and improve graft survival (Seo SK et al.,(2004)Nature Med.10(10):1088-1094;Sun Y et al.,(2002)J.Immunol.168(3):1457-1465;Agarwal A et al.,(2008)Curr.Opin.Organ.Transplant 13(4):366-372).

[0006] In particular, the effects of 4-1BB have been studied in greater detail in cancer than in other pathologies. 4-1BB signaling was found to disrupt and reverse established anergy in cytotoxic T lymphocytes. For example, in the B16.SIY melanoma model, administration of anti-4-1BB mAb restored the function of CD8+ TILs that had lost their ability to secrete IL-2 (Cariad Chester et al.,(2018)supra). Most of the antitumor effects of anti-4-1BB mAb are exhibited by CD8+ T cells, but NK, NKT cells, dendritic cells, and CD4+ T cells have also proven to be important and necessary for tumor therapy (Dass SV et al.,(2014)BMB Rep.47(3):122-129). Furthermore, upregulation of some adhesion molecules, such as ICAM-1 and CXAM-1, following anti-4-1BB administration was found to increase T cell migration to tumor sites (Palazon A et al.,(2011)Cancer Res.71(3):801-811). More importantly, the efficacy of anti-4-1BB therapy has also been demonstrated in preliminary models of other solid tumors and lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, melanoma, ovarian cancer, and squamous cell lung cancer (Cariad Chester et al.,(2018)supra).

[0007] Urelumab is a fully human IgG4 monoclonal antibody developed by BMS and was the first anti-4-1BB therapeutic to enter clinical trials. It specifically binds to and activates 4-1BB-expressing immune cells and showed promising efficacy in phase 1 and 2 monotherapy trials, but showed hepatotoxicity in a dose-dependent manner. Urelumab monotherapy and combination therapy at tolerated doses showed promising immune stimulatory pharmacodynamic effects but limited antitumor efficacy. Utomilumab, a humanized IgG2 monoclonal antibody from Pfizer, has shown a good safety profile but is a weaker 4-1BB agonist compared to urelumab. It is currently undergoing clinical trials in combination with rituximab, pembrolizumab, Keytruda, avelumab, and other antitumor antibodies for the treatment of various cancers (Cariad Chester et al., (2018) supra).

[0008] In addition, combination therapy of anti-4-1BB with other antibodies (such as anti-PD-1 antibodies) is also in clinical trials. For example, bispecific antibodies are needed that simultaneously target both PD-L1 and 4-1BB to block the PD-1 / PD-L1 pathway and provide costimulatory signals via 4-1BB upon crosslinking of PD-L1 by PD-L1-expressing tumor cells. However, the bispecific antibodies described in US Patent US2017 / 0198050A1 activate or induce 4-1BB signaling without crosslinking with target cells, raising concerns about potential toxicity caused by 4-1BB at high concentrations.

[0009] Therefore, there is still a strong need in the art to develop new anti-4-1BB antibodies with an optimal balance between safety profile and agonism. Such anti-4-1BB antibodies will also be useful for constructing new bispecific antibodies, including bispecific antibodies that can bind to 4-1BB and other antigens, such as PD-L1. Such bispecific antibodies that can specifically bind to 4-1BB while also binding to other antigens, such as PD-L1 with higher affinity, are also the object of development in the field of 4-1BB antibodies. Summary of the Invention

[0010] The present disclosure provides isolated monoclonal agonistic antibodies, e.g., human, murine, chimeric or humanized monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to 4-1BB (e.g., human 4-1BB). In one embodiment, the antibodies of the present invention provide an optimal balance between safety profile and agonism.

[0011] The antibodies or antigen-binding portions thereof of the present disclosure can be used in a variety of applications, including detection of 4-1BB protein and treatment of 4-1BB-associated diseases, such as tumors, infectious diseases and autoimmune diseases.

[0012] Accordingly, in one aspect, the disclosure relates to an isolated monoclonal antibody (e.g., a human antibody) or antigen-binding portion thereof that binds to 4-1BB, having a heavy chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, the CDR2 region, and the CDR3 region comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) SEQ ID NOs: 1, 2, and 3, respectively; (2) SEQ ID NOs: 7, 8, and 9, respectively; (3) SEQ ID NOs: 13, 14, and 15, respectively; or (4) SEQ ID NOs: 19, 20, and 21, respectively.

[0013] In one embodiment, an isolated monoclonal antibody or antigen-binding portion thereof of the disclosure comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25, 27, 29, 31, or 77, wherein the antibody or antigen-binding fragment thereof binds to 4-1BB.

[0014] In one embodiment, an isolated monoclonal antibody or antigen-binding portion thereof of the present disclosure that binds to 4-1BB comprises a light chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein said CDR1 region, CDR2 region, and CDR3 region comprise amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) SEQ ID NOs: 4, 5, and 6, respectively; (2) SEQ ID NOs: 10, 11, and 12, respectively; (3) SEQ ID NOs: 16, 17, and 18, respectively; or (4) SEQ ID NOs: 22, 23, and 24, respectively.

[0015] In one embodiment, an isolated monoclonal antibody or antigen-binding portion thereof of the disclosure comprises a light chain variable region comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26 (X1=S or G), 28, 30, 32 or 78, wherein the antibody or antigen-binding fragment thereof binds to 4-1BB.

[0016] In one embodiment, an isolated monoclonal antibody or antigen-binding portion thereof of the present disclosure comprises a heavy chain variable region and a light chain variable region, each of which comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1, CDR2 and CDR3 of the heavy chain variable region and the CDR1, CDR2 and CDR3 of the light chain variable region are set forth in (1) SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively; (2) SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively; (3) or (4) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively, or SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively, wherein the antibody or antigen-binding fragment thereof binds to 4-1BB.

[0017] In one embodiment, an isolated monoclonal antibody or antigen-binding portion thereof of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 25 and 26, respectively (X1=S); (2) SEQ ID NOs: 25 and 26, respectively (X1=G); (3) SEQ ID NOs: 27 and 28, respectively; (4) SEQ ID NOs: 29 and 30, respectively; (5) SEQ ID NOs: 31 and 32, respectively; or (6) SEQ ID NOs: 77 and 78, respectively, wherein the antibody or antigen-binding fragment thereof binds to 4-1BB.

[0018] In one embodiment, an isolated monoclonal antibody or antigen-binding portion thereof of the present disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region, wherein the heavy chain constant region can be a human IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region can be a human lambda or kappa constant region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth above, wherein the antibody or antigen-binding fragment thereof binds to 4-1BB. In a further embodiment, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 33. In a further embodiment, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 35 or 63. The heavy chain constant region may be any other suitable constant region, such as a constant region derived from a human IgG constant region, for example an IgG4 constant region with the mutation S228P having the amino acid sequence set forth in SEQ ID NO: 75, or an IgG1 constant region with the mutations L234A, L235A, D265A, P329A (EU numbering) having the amino acid sequence set forth in SEQ ID NO: 34. The light chain constant region may be any other suitable constant region derived from a human kappa or lambda constant region.

[0019] The antibody of the present disclosure, in some embodiments, comprises or consists of two heavy chains and two light chains connected by disulfide bonds, where each heavy chain comprises the heavy chain constant region, the heavy chain variable region or the CDR sequence, and each light chain comprises the light chain constant region, the light chain variable region or the CDR sequence, where the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. The antibody of the present disclosure may be a full-length antibody, e.g., an IgG1, IgG2 or IgG4 isotype. The antibody of the present disclosure may comprise a kappa or lambda constant region. The antibody or antigen-binding portion thereof according to other embodiments of the present disclosure may be a single chain variable fragment (scFv) antibody, or an antibody fragment, such as a Fab or Fab'2 fragment.

[0020] Exemplary antibodies or antigen-binding portions thereof of the present disclosure are agonistic anti-4-1BB antibodies that specifically bind to 4-1BB, particularly human 4-1BB, and activate 4-1BB signaling.

[0021] The present disclosure also provides bispecific molecules comprising an antibody or antigen-binding portion thereof of the present disclosure linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than the antibody or antigen-binding portion thereof. The bispecific molecule may bind to 4-1BB and another molecule, such as PD-1, PD-L1 or CTLA-4, preferably, the molecule is PD-L1.

[0022] In one aspect, the antibody of the present disclosure refers to a bispecific antibody. Thus, the present disclosure also provides a bispecific antibody comprising a first binding region that specifically binds to 4-1BB (e.g., human 4-1BB) and a second binding region that specifically binds to a second antigen associated with cancer, an infectious disease, or an autoimmune disease. In one embodiment, the second antigen is selected from (human) PD-1, PD-L1, or CTLA-4. More preferably, the second antigen is (human) PD-L1. In one embodiment, the first binding region that specifically binds to 4-1BB comprises a heavy chain variable region as described above and / or a light chain variable region as described above. In one embodiment, the first binding region comprises the CDR1 region, CDR2 region, and CDR3 region of the heavy chain variable region as described above, and / or the CDR1 region, CDR2 region, and CDR3 region of the light chain variable region as described above. In one embodiment, the first binding region specifically binds to (human) 4-1BB and specifically binds to the second antigen with high affinity.

[0023] In one embodiment, the bispecific antibody comprises a first binding region that specifically binds human 4-1BB and a second binding region that specifically binds human PD-L1 with high affinity, such that the bispecific antibody is active only in a tumor microenvironment that has PD-L1 expression, while avoiding systemic stimulation of T cells.

[0024] Also provided is a composition comprising an antibody or antigen-binding portion thereof, or a bispecific molecule of the present disclosure and a pharma- ceutically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition.

[0025] The present disclosure also includes a nucleic acid molecule encoding the antibody or antigen-binding portion thereof or a chain of the antibody of the present disclosure, as well as an expression vector comprising the nucleic acid, and a host cell comprising the expression vector or having its genome integrated with a polynucleotide encoding the antibody or antigen-binding portion thereof. Also provided is a method for preparing an anti-4-1BB antibody using a host cell comprising an expression vector, the method comprising (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell or a cell culture thereof.

[0026] In yet another aspect, the present disclosure provides a method of treating a cancer disease in a subject, the method comprising administering to a subject in need thereof an antibody or antigen-binding portion thereof of the present disclosure. In some embodiments, the method comprises administering a composition, or a bispecific molecule, or optionally a nucleic acid molecule capable of expressing the composition or bispecific molecule in a subject, of the present disclosure. The bispecific molecule can bind to 4-1BB and another protein, such as PD-1, PD-L1 or CTLA-4, preferably PD-L1. In some embodiments, at least one additional antibody may be administered together with the antibody or antigen-binding portion thereof of the present disclosure, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In some embodiments, at least one additional anti-cancer agent may be administered together with the antibody or antigen-binding portion thereof of the present disclosure. The cancer may be a solid or non-solid cancer, for example, a cancer of the digestive tract (gastrointestinal tract), such as colon cancer, colorectal cancer (large intestine cancer), or rectal cancer.

[0027] In another aspect, the present disclosure discloses a method for treating an infectious disease in a subject, the method comprising administering to a subject in need thereof an antibody, antigen-binding portion thereof, specific molecule, or pharmaceutical composition of the present disclosure, optionally together with an additional antiviral, antibacterial, or antifungal agent.

[0028] In another aspect, the present disclosure discloses a method for treating an autoimmune disease, comprising administering to a subject in need thereof an antibody, an antigen-binding portion thereof, a specific molecule, or a pharmaceutical composition of the present disclosure. The autoimmune disease may be asthma or rheumatoid arthritis. Optionally, an additional anti-asthma or anti-rheumatoid arthritis agent may be administered.

[0029] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. [Brief description of the drawings]

[0030] [Figure 1A] FIG. 1 shows the binding ability of anti-41BB antibodies 41BB-2-IgG2 (A), 41BB-9-IgG2 (B), 41BB-13-IgG2 (C), and 41BB-27-IgG2 (D) to human 4-1BB in an ELISA assay. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above.

[0031] [Figure 2A] FIG. 1 shows the binding ability of anti-4-1BB antibodies 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2 (A) and 41BB-27-IgG2 (B) to human CD40, human HVEM, human OX40, human CD27, human GITR and Rhesus 4-1BB in an ELISA assay. [Figure 2B] Same as above.

[0032] [Figure 3A] FIG. 13 shows the agonistic activity of anti-4-1BB antibodies 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2 and 41BB-27-IgG2 in 4-1BB signaling with or without cross-linking. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above.

[0033] [Figure 4A] FIG. 13 shows the agonistic activity of anti-4-1BB antibodies 41BB-2-IgG1, 41BB-9-IgG1, 41BB-13-IgG1 and 41BB-27-IgG4 in 4-1BB signaling with or without a cross-linker. [Figure 4B] Same as above.

[0034] [Figure 5A] FIG. 1 shows the structures of bispecific antibodies P4B-3 (A) and P4B-2 (B). [Figure 5B] Same as above.

[0035] [Figure 6] Figure 2 shows bispecific antibody binding to human PD-L1 in an ELISA assay.

[0036] [Figure 7] FIG. 1 shows binding of bispecific antibodies to human 4-1BB in an ELISA assay.

[0037] [Figure 8]Figure 1 shows the ability of bispecific antibodies (P4B-3 and NM21-PRO1186) to induce PD-L1 binding-dependent 4-1BB stimulation in a 4-1BB reporter gene assay using target CHO-K1-PDL1 cells.

[0038] [Figure 9A] FIG. 1 shows the ability of bispecific antibodies (P4B-3 and INBRX-105-1) to induce 4-1BB stimulation in a 4-1BB reporter gene assay with or without target cells A375 / PD-L1. [Figure 9B] Same as above.

[0039] [Figure 10] FIG. 1 shows that P4B-3 activates human PBMC to release IL-2.

[0040] [Figure 11] FIG. 1 shows tumor growth curves after treatment.

[0041] [Figure 12] FIG. 1 shows the percentage of mCD3+mCD8+ T cells in the tumor microenvironment (TME).

[0042] [Figure 13] FIG. 1 shows the percentage of mCD4 mTregs in the TME. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0044] The term "4-1BB" refers to member 9 of the tumor necrosis factor receptor superfamily. The term "4-1BB" includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human 4-1BB protein may in certain cases cross-react with 4-1BB protein from species other than human, such as monkeys. In other embodiments, an antibody specific for human 4-1BB protein may be completely specific for human 4-1BB protein and not cross-reactive with other species or types, or may cross-react with 4-1BB from certain other species, but not from all other species.

[0045] The term "human 4-1BB" refers to a 4-1BB protein having an amino acid sequence of human origin, such as the amino acid sequence of human 4-1BB having GenBank Accession No. NP_001552.2. In one embodiment, human 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 39. The terms "monkey or Rhesus 4-1BB" and "mouse 4-1BB" refer to monkey and mouse 4-1BB sequences, respectively, such as those having the amino acid sequences having GenBank Accession Nos. NP_001253057.1 and NP_033430.1, respectively.

[0046] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. Furthermore, as used herein, the term "antibody" also includes multispecific antibodies, such as bispecific or trispecific antibodies. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region is composed of C H1 , C H2 and C H3 Each light chain is composed of three domains: L ) and a light chain constant region (herein abbreviated as C L The light chain constant region is composed of CL It consists of one domain: V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0047] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., 4-1BB protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; and (iii) a V H and C H1 (iv) a single-armed V L and V H (v) an Fv fragment consisting of a V H (vi) a dAb fragment consisting of a heavy chain variable region containing a single variable domain and two constant domains (Ward et al., (1989) Nature 341:544-546); ... L and V Hare encoded by separate genes, but can be linked by synthetic linkers that allow them to be made into a single protein chain by recombinant methods. L and V H can pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are intended to be encompassed within the term "antigen-binding portion" of an antibody. These fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0048] The term "bispecific" means that an antibody can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antibody contains two antigen-binding regions, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two separate cells. As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a stretch of consecutive amino acids or a conformational configuration composed of different regions of non-consecutive amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0049] The term "antigen-binding region" as used herein refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding region can direct the entity to which it is attached (e.g., a second antigen-binding region) to a target site, e.g., a specific type of tumor cell bearing the antigenic determinant. In another embodiment, an antigen-binding region (e.g., a first antigen-binding region) can activate signal transduction through its target antigen, e.g., a T cell receptor antigen. Antigen-binding regions include antibodies or antigen-binding fragments thereof as defined herein. Particular antigen-binding regions include antigen-binding fragments of antibodies that include an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding region may include an antibody constant region as defined herein and known in the art.

[0050] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding region to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) (analyzed on a BIAcore instrument).

[0051] Unless otherwise indicated, the term "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding bearer (e.g., a monoclonal antibody and an antigen, an antigen-binding region and an antigen, or a receptor and its ligand). Affinity can be measured by well-established methods known in the art, including the methods described herein.

[0052] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to 4-1BB protein is substantially free of antibodies that specifically bind to antigens other than 4-1BB protein). However, an isolated antibody that specifically binds to human 4-1BB protein may have cross-reactivity to other antigens (e.g., 4-1BB proteins from other species). Furthermore, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0053] The term "human antibody" as used herein is intended to include antibodies having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species are grafted onto human framework sequences.

[0054] The term "agonistic 4-1BB antibody" or "agonistic anti-4-1BB antibody" refers to an anti-4-1BB antibody that binds to 4-1BB and activates or induces 4-1BB signaling to promote activation and / or proliferation of immune cells such as T cells.

[0055] The term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0056] The terms "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0057] As used herein, an antibody that "specifically binds to human 4-1BB" is intended to refer to an antibody that binds to human 4-1BB protein (and optionally 4-1BB proteins from one or more non-human species) but does not substantially bind to non-4-1BB proteins. D is 1.0×10 -6 M or less, preferably 5.0×10 -7 M or less, preferably 1.0×10 -7 It is preferable that the 4-1BB protein binds to the human 4-1BB protein with an M of 0.1 or less.

[0058] As used herein, the term "does not substantially bind" to a protein or cell means that the antibody does not specifically bind to the protein or cell or does not bind with high affinity, i.e., does not bind to the protein or cell. D is 1.0×10 -6 M or more, preferably 1.0×10 -5 M or more, preferably 1.0×10 -4 M or more, and more preferably 1.0×10 -3 M or more, and even more preferably 1.0×10 -2 It means that the molecule binds to the protein or cell at M or more.

[0059] The term "specifically binds to a second antigen (PD-L1) with high affinity" for an IgG antibody or antigen-binding region thereof means K D is 1.0×10 -8 M or less, preferably 5.0×10 -9 M or less, and even more preferably 1.0×10 -9 This refers to an antibody or antigen-binding region that is equal to or smaller than M.

[0060] As used herein, the term "K assoc " or "K a " is intended to refer to the association rate of a particular antibody-antigen interaction. dis " or "K d " is intended to refer to the off-rate of a particular antibody-antigen interaction. D " is the dissociation constant, Ka K for d The ratio of (i.e., K d / K a ) and expressed as molar concentration (M). D Values ​​can be determined using methods well established in the art. Preferably, the K D The determination of is preferably performed using surface plasmon resonance or, more preferably, a biosensor system (e.g., Biacore TM This is done using the system.

[0061] The term “EC 50 " is known as the half-maximal effective concentration and refers to the concentration of antibody that elicits a response halfway between the baseline and maximum after a specific exposure time.

[0062] The term “IC 50 " Also known as the half-maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% compared to the absence of the antibody.

[0063] The term "subject" includes humans and non-human animals. The term "non-human animals" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0064] The term "therapeutically effective amount" refers to an amount of an antibody of the present disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in relation to the condition being treated, where the actual effective amount will be readily discerned by one of skill in the art.

[0065] As used herein, the term "therapeutic agent" includes substances effective in the prevention or treatment of neoplasia (e.g., cancer) or infectious or autoimmune diseases, including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against immune checkpoint molecules), anti-infective agents, immunomodulatory agents, and small molecule drugs.

[0066] The term "chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer.

[0067] The term "anti-infective agent" includes molecules that specifically inhibit or eliminate the growth of microorganisms, such as viruses, bacteria, fungi, or protozoans, e.g., parasites, at dosage concentrations and intervals that are not lethal to the host.

[0068] As used herein, the term "anti-infective agent" includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In one particular embodiment, the anti-infective agent is non-toxic to the host at the concentrations and intervals of administration.

[0069] Immunomodulatory agents include immune checkpoint molecule inhibitors and costimulatory molecule activators.

[0070] The term "small molecule drug" refers to a low molecular weight organic compound that can regulate biological processes. "Small molecule" is defined as a molecule with a molecular weight usually less than 2 kD, preferably less than 1 kD, more preferably less than about 500 Daltons. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics.

[0071] The terms "cancer" and "cancerous" refer to or describe the physiological disorder in mammals that is typically characterized by unregulated cell growth (cell proliferation).

[0072] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

[0073] The term "infectious disease" refers to a disease caused by a pathogen, including protozoa, such as, for example, a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

[0074] The term "tumor immune evasion" refers to tumors that evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" and when evasion is weakened, tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0075] Various aspects of the disclosure are described in further detail in the following subsections.

[0076] An anti-4-1BB antibody having binding affinity for human 4-1BB.

[0077] Exemplary antibodies or antigen-binding portions thereof of the present disclosure specifically bind to human 4-1BB. Exemplary antibodies or antigen-binding portions thereof of the present disclosure are agonistic antibodies that activate or induce 4-1BB signaling to promote activation and / or proliferation of immune cells, such as T cells.

[0078] In one embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof, comprising three heavy chain variable region CDRs (CDR1, CDR2 and CDR3). In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof, comprising three light chain variable region CDRs (CDR1, CDR2 and CDR3). In a further embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof comprising three heavy chain variable region CDRs (CDR1, CDR2 and CDR3) and three light chain variable region CDRs (CDR1, CDR2 and CDR3).

[0079] In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof, comprising a heavy chain variable region. In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof, comprising a light chain variable region. In a further embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region. In one embodiment, the heavy chain variable region comprises three heavy chain variable region CDRs (CDR1, CDR2 and CDR3). In another embodiment, the light chain variable region comprises three light chain variable region CDRs (CDR1, CDR2 and CDR3).

[0080] In one embodiment, the three heavy chain variable regions CDR1, CDR2 and CDR3 are (i) CDR1, CDR2 and CDR3 derived from a heavy chain variable region VH comprising or consisting of the sequence set forth in SEQ ID NO: 25, 27, 29, 31 or 77; or (ii) CDR1, CDR2 and CDR3 of (i), further comprising a total of from one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of (i). In one embodiment, the three light chain variable regions CDR1, CDR2 and CDR3 are (i) CDR1, CDR2 and CDR3 derived from a light chain variable region VL comprising or consisting of the sequence set forth in SEQ ID NO: 26, 28, 30, 32 or 78; or (ii) CDR1, CDR2 and CDR3 of (i), further comprising a total of from one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of (i). In one embodiment, the present invention relates to a heavy chain variable region V consisting of the sequence set forth in SEQ ID NO: 25, 27, 29, 31 or 77. H and a light chain variable region V consisting of the sequence shown in SEQ ID NO: 26, 28, 30, 32 or 78. L and three light chain variable regions CDR1, CDR2 and CDR3 derived from the antibody or antigen-binding fragment thereof. In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof comprising: (1) three CDRs (CDR1, CDR2, and CDR3) derived from a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 25 or 77, and three CDRs (CDR1, CDR2, and CDR3) derived from a light chain variable region consisting of the sequence shown in SEQ ID NO: 26 or 78; (2) three CDRs (CDR1, CDR2, and CDR3) derived from a heavy chain variable region consisting of the sequence set forth in SEQ ID NO: 27, and three CDRs (CDR1, CDR2, and CDR3) derived from a light chain variable region consisting of the sequence set forth in SEQ ID NO: 28; (3) three CDRs (CDR1, CDR2, and CDR3) derived from a heavy chain variable region consisting of the sequence set forth in SEQ ID NO: 29, and three CDRs (CDR1, CDR2, and CDR3) derived from a light chain variable region consisting of the sequence set forth in SEQ ID NO: 30; (4) Three CDRs (CDR1, CDR2, and CDR3) derived from a heavy chain variable region consisting of the sequence set forth in SEQ ID NO: 31, and three CDRs (CDR1, CDR2, and CDR3) derived from a light chain variable region consisting of the sequence set forth in SEQ ID NO: 32.

[0081] In one embodiment, V H CDR1 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 13 or 19, or H CDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 13 or 19. In one embodiment, V H CDR2 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 8, 14 or 20, or HCDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 8, 14 or 20. In one embodiment, V H CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 9, 15 or 21, or H The CDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 9, 15 or 21. In one embodiment, V L CDR1 of comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16 or 22, or L CDR1 of comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16 or 22. In one embodiment, V L CDR2 of comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, 17 or 23, or L CDR2 of comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, 17 or 23. In one embodiment, V L CDR3 of comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 12, 18 or 24, or L The CDR3 of comprises or consists of an amino acid sequence having only one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 18 or 24.

[0082] In one embodiment, three heavy chain variable regions V H CDR1, CDR2 and CDR3 are (i) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively; or (ii) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 7, 8 and 9, respectively; or (iii) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 13, 14 and 15, respectively; or (iv) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively; or (v) CDR1, CDR2 and CDR3 of any of (i) to (iv), further comprising a total of one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of any of (i) to (iv). In one embodiment, three light chain variable regions V L CDR1, CDR2 and CDR3 of (i) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively; or (ii) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 10, 11 and 12, respectively; or (iii) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; or (iv) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively; or (v) CDR1, CDR2 and CDR3 of any of (i) to (iv), further comprising a total of one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of any of (i) to (iv). In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof comprising: (i) V consisting of the sequences shown in SEQ ID NOs: 1, 2 and 3, respectively H CDR1, CDR2 and CDR3, and V consisting of the sequences shown in SEQ ID NOs: 4, 5 and 6, respectively L CDR1, CDR2 and CDR3 of; (ii) V consisting of the sequences shown in SEQ ID NOs: 7, 8 and 9, respectively H CDR1, CDR2 and CDR3, and V consisting of the sequences shown in SEQ ID NOs: 10, 11 and 12, respectively L CDR1, CDR2 and CDR3; (iii) V consisting of the sequences shown in SEQ ID NOs: 13, 14 and 15, respectively. H CDR1, CDR2 and CDR3, and V consisting of the sequences shown in SEQ ID NOs: 16, 17 and 18, respectively L CDR1, CDR2 and CDR3; or (iv) V consisting of the sequences shown in SEQ ID NOs: 19, 20 and 21, respectively. H CDR1, CDR2 and CDR3, and V consisting of the sequences shown in SEQ ID NOs: 22, 23 and 24, respectively L CDR1, CDR2 and CDR3.

[0083] In one embodiment, the heavy chain variable region comprises: (i) comprises or consists of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 27, 29, 31 or 77; or (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 27, 29, 31, or 77; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 27, 29, 31 or 77, preferably, said amino acid modifications do not occur in a CDR region, more preferably, said amino acid modifications occur in a FR region, such as the FR1, FR2, FR3 or FR4 region. In another embodiment, the light chain variable region is (i) comprises or consists of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 30, 32 or 78; or (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 30, 32, or 78; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 30, 32 or 78, preferably said amino acid modifications do not occur in the CDR regions, more preferably said amino acid modifications occur in the FR regions, e.g. the FR1 region, the FR2 region, the FR3 region or the FR4 region. In one embodiment, the variable region can be modified to improve purification of the antibody, for example, at the end of the heavy chain variable region, an amino acid can be mutated from S to G to obtain an antibody that can be prepared with high purity. In another embodiment, the variable region can be modified to improve the stability of the antibody or antigen-binding portion (e.g., scFv). For example, a mutation can be made to form a disulfide between the heavy chain variable region and the light chain variable region. For example, the mutation in the heavy chain variable region is G44C (Eu numbering) and / or the mutation in the light chain variable region is T104C (Eu numbering). In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25, 27, 29, 31 or 77; and / or A light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 26, 28, 30, 32 or 78. In another embodiment, the present invention relates to an anti-4-1BB antibody or antigen-binding fragment thereof comprising: (1) a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:26; (2) a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:27, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:28; (3) a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:30; (4) a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 31, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 32; or (5) A heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 78.

[0084] In a further embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, and / or a light chain constant region.

[0085] In one embodiment, the heavy chain constant region is or is derived from a human IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region, preferably an IgG1, IgG2 or IgG4 constant region. In another embodiment, the heavy chain constant region comprises: (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 33, 34 or 75; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33, 34 or 75; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence set forth in SEQ ID NO: 33, 34 or 75. In one embodiment, the light chain constant region is or is derived from a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region, e.g., a human lambda light chain constant region. In another embodiment, the light chain constant region comprises: (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 35 or 63; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 35 or 63; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence set forth in SEQ ID NO: 35 or 63.

[0086] In one embodiment, the constant region of the antibody can be mutated to improve the production and purification of the antibody. For example, the IgG4 constant region can have the mutation S228P (EU numbering). The IgG1 constant region can have the mutations L234A, L235A, D265A, P329A (Eu numbering).

[0087] In one embodiment, the antigen-binding fragment of the antibody of the present disclosure is an scFv fragment. In particular, the scFv fragment comprises a light chain variable region and a heavy chain variable region linked by a linker. In one embodiment, the linker is a flexible linker, such as a linker having glycine and / or serine residues, alone or in combination. In one embodiment, the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, where n is a positive integer of 1 or more, for example, n is a positive integer of 1 to 7, for example, n is 2, 3, 4, 5, 6. In one embodiment, n is 1, 2, 3, or 4. In a particular embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:52.

[0088] Preferred antibodies of the present disclosure are monoclonal antibodies.

[0089] Monoclonal anti-4-1BB antibody

[0090] The antibody of the present disclosure may be a monoclonal antibody that is structurally and chemically characterized as described below and in the following examples. The amino acid sequence numbers of the CDR regions or heavy / light chain variable regions of the antibody are summarized in Table 1 below. The heavy chain constant region of the antibody may be, for example, a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region or a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 33, 34 or 75, and the light chain constant region of the antibody may be, for example, a human lambda constant region having the amino acid sequence shown in SEQ ID NO: 35 or 63. The antibody comprises or consists of two heavy chains and two light chains linked by disulfide bonds, and the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. [Table 1]

[0091] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined by other systems, such as Chothia, and IMGT, AbM, or Contact numbering systems / methods, based on the heavy / light chain variable region sequences. It should be noted that the boundaries of the CDRs of the variable regions of antibodies obtained with different numbering systems may be different. That is, the CDR sequences of the variable regions of antibodies defined by different numbering systems are different. Thus, when an antibody is defined by a specific CDR sequence defined by the present invention, the scope of the antibody also includes antibodies whose variable region sequences comprise that specific CDR sequence, but whose CDR boundaries are claimed to be different from the specific CDR boundaries defined by the present invention due to the application of different protocols (e.g., different numbering system rules or combinations thereof).

[0092] V of other anti-4-1BB antibodies that bind to human 4-1BB H and V LThe sequence (or CDR sequence) of the anti-4-1BB antibody of the present disclosure H and V L Preferably, the V H Chain and V L When chains (or CDRs within such chains) are mixed and matched, a particular V H / V L V from pairing H The sequences were then divided into structurally similar V H Similarly, preferably, a particular V H / V L V from pairing L The sequences were then divided into structurally similar V L Thus, in one embodiment, an antibody or antigen-binding portion thereof of the present disclosure comprises: (a) a heavy chain variable region comprising an amino acid sequence listed in Table 1 above; and (b) a light chain variable region comprising an amino acid sequence listed in Table 1 above, or the V of another anti-4-1BB antibody. L Including, wherein said antibody specifically binds to human 4-1BB.

[0093] In another embodiment, an antibody or antigen-binding portion thereof of the present disclosure comprises: (a) the CDR1, CDR2 and CDR3 regions of a heavy chain variable region listed in Table 1 above; and (b) comprising the CDR1, CDR2 and CDR3 regions of a light chain variable region listed in Table 1 above, or the CDRs of another anti-4-1BB antibody; wherein said antibody specifically binds to human 4-1BB.

[0094] In yet another embodiment, the antibody or antigen-binding portion thereof comprises the CDR2 of the heavy chain variable region of an anti-4-1BB antibody combined with the CDR1 and / or CDR3 of the heavy chain variable region, and / or the CDR1, CDR2 and / or CDR3 of the light chain variable region of a different anti-4-1BB antibody.

[0095] Furthermore, as is well known in the art, the CDR3 domain can alone, independently of the CDR1 and / or CDR2 domains, determine the binding specificity of an antibody to a cognate antigen, allowing predictable generation of multiple antibodies with the same binding specificity based on a common CDR3 sequence. For example, Klimka et al.,British J.of Cancer 83(2):252-260(2000);Beiboer et al.,J.Mol.Biol.296:833-849(2000);Rader et al.,Proc.Natl.Acad.Sci.USA95:8910-8915(1998);Barbas et al. al.,J.Am.Chem.Soc.116:2161-2162(1994);Barbas et al.,Proc.Natl.Acad.Sci.USA92:2529-2533 (1995);Ditzel et al.,J.Immunol.157:739-749(1996);Berezov et al.,BIAjournal 8:Scientific Review 8(2001);Igarashi et al. See, e.g., Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185, each of which is incorporated herein by reference in its entirety.

[0096] Thus, in another embodiment, the antibody of the present disclosure comprises CDR2 of the heavy chain variable region of an anti-4-1BB antibody and at least CDR3 of the heavy and / or light chain variable region of an anti-4-1BB antibody, or CDR3 of the heavy and / or light chain variable region of another anti-4-1BB antibody that can specifically bind to human 4-1BB. These antibodies preferably (a) compete with the anti-4-1BB antibody of the present invention for binding to 4-1BB, (b) retain functional properties, (c) bind to the same epitope, and / or (d) have similar binding affinity. In yet another embodiment, the antibody may further comprise CDR2 of the light chain variable region of an anti-4-1BB antibody, or CDR2 of the light chain variable region of another anti-4-1BB antibody that can specifically bind to human 4-1BB. In another embodiment, an antibody of the present invention may comprise CDR1 of the heavy and / or light chain variable region of an anti-4-1BB antibody, or CDR1 of the heavy and / or light chain variable region of another anti-4-1BB antibody capable of specifically binding to human 4-1BB.

[0097] A preferred antibody of the present disclosure is a bispecific antibody.

[0098] bispecific molecules

[0099] In other aspects, the present disclosure features bispecific molecules comprising one or more antibodies or antibody-binding fragments of the present disclosure linked to at least one other functional molecule, such as other peptides or proteins (e.g., another antibody and a ligand for a receptor) that produce a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules with three or more specificities.

[0100] In one embodiment, the bispecific molecule has a second specificity in addition to the anti-4-1BB binding specificity, which may be for PD-1, PD-L1 or CTLA-4.

[0101] In one embodiment, the bispecific molecule has a third specificity in addition to the anti-4-1BB binding specificity and the anti-PD-L1 binding specificity, which may be for PD-1 or CTLA-4 for cancer treatment.

[0102] Bispecific molecules come in many different formats and sizes. In the size spectrum, bispecific molecules retain the traditional antibody format, except that instead of having two binding arms with the same specificity, they have two binding arms, each with a different specificity. At the other extreme, a bispecific molecule consists of two single-chain antibody fragments (scFv's) linked by a peptide chain, the so-called Bs(scFv)2 construct. Intermediate size bispecific molecules include two different F(ab) fragments linked by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, and chemical methods. See, for example, Kufer et al, cited supra, Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998), and van Spriel et al., Immunology Today, 21(8), 391-397 (2000). These references are incorporated herein.

[0103] In one embodiment, the bispecific molecule of the present invention is a bispecific antibody comprising a first binding region that specifically binds to 4-1BB and a second binding region that specifically binds to a second antigen associated with cancer, an infectious disease, or an autoimmune disease. Bispecific antibodies can have several structural formats (cf, Aran F. Labrijn et al., Bispecific antibodies: a mechanistic review of the pipeline, Nature Reviews Drug Discovery, volume 18, pages 585-608 (2019)). For example, bispecific antibodies can be IgG-like antibodies, i.e., full-length bispecific antibodies, or non-IgG-like bispecific antibodies that are not full-length antibody constructs.

[0104] In particular, the second antigen is selected from PD-1, PD-L1 or CTLA-4, e.g., human PD-1, PD-L1 or CTLA-4. More preferably, the second antigen is PD-L1, e.g., human PD-L1. In one embodiment, the human PD-L1 comprises an amino acid sequence set forth in SEQ ID NO:61, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:61. In another embodiment, human PD-L1 is encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:62, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:62.

[0105] In one embodiment, the first binding region comprises a V H and / or V LIn another embodiment, the first binding region comprises the heavy chain variable region CDR1, CDR2 and / or CDR3, and / or the light chain variable region CDR1, CDR2 and / or CDR3, which are disclosed for the anti-4-1BB antibody or antibody binding fragment as described above. In a further embodiment, the first binding region further comprises a constant region, e.g., a heavy chain constant region or a light chain constant region, which are disclosed for the anti-4-1BB antibody or antibody binding fragment as described above. In one embodiment, the first binding region comprises an scFv of the anti-4-1BB antibody disclosed herein above.

[0106] In another embodiment, the second binding domain comprises a V-binding domain as described in the art for an anti-PD-L1, or anti-PD-1 or anti-CTLA-4 antibody or antibody binding fragment. H and / or V L In another embodiment, the second binding region comprises a heavy chain variable region CDR1, CDR2 and / or CDR3, and / or a light chain variable region CDR1, CDR2 and / or CDR3, which are disclosed for an anti-PD-L1, or anti-PD1 or anti-CTLA-4 antibody or antibody binding fragment in the art. In one embodiment, the second binding region further comprises a constant region, e.g., a heavy chain constant region and / or a light chain constant region, e.g., those disclosed in the present disclosure.

[0107] In certain embodiments, the second binding region is capable of binding to human PD-L1 and is selected from the group consisting of V and VL1 domains disclosed for anti-PD-L1 antibodies or antibody-binding fragments in the art. H and / or V L In another embodiment, the second binding region comprises a heavy chain variable region CDR1, CDR2 and / or CDR3, and / or a light chain variable region CDR1, CDR2 and / or CDR3, which have been disclosed for an anti-PD-L1 antibody or antibody-binding fragment in the art. In one embodiment, the second binding region further comprises a constant region, e.g., a heavy chain constant region and / or a light chain constant region.

[0108] For the construction of bispecific antibodies, the V of the bispecific antibody H and / or V L V of the monoclonal antibodies from which they are derived H and / or V L It is known that one or several amino acids may differ from the V H and V L can have one or more mutations to make the scFv more stable. H and / or V L can have mutations to form disulfide bonds with each other to make the antibody or antigen-binding portion thereof more stable. For example, V H has G44C (EU numbering), and V L may have T104C (EU numbering).

[0109] In certain embodiments, the bispecific antibody comprises the following chains: (1) Chain 1: a heavy chain of an antibody against a second antigen linked at the N-terminus or C-terminus, with or without a linker, to an scFv fragment of an anti-4-1BB antibody of the present disclosure: and (2) Chain 2: The light chain of an antibody against a second antigen. In certain embodiments, a bispecific antibody comprises two chains 1 and two chains 2 linked by a disulfide bond. In further particular embodiments, the structure of a bispecific antibody is shown in Figure 5A or Figure 5B. In particular, the heavy chain of the antibody against the second antigen may be any of the V and VIII heavy chains disclosed in the art for anti-PD-L1, or anti-PD-1 or anti-CTLA-4 antibodies or antibody-binding fragments. H In another embodiment, the heavy chain of the antibody against the second antigen comprises the heavy chain variable regions CDR1, CDR2 and / or CDR3 disclosed in the art for anti-PD-L1, or anti-PD-1 or anti-CTLA-4. In particular, the light chain of the antibody against the second antigen comprises the VDR1, CDR2 and / or CDR3 disclosed in the art for anti-PD-L1, or anti-PD-1 or anti-CTLA-4 antibodies or antibody binding fragments.L In another embodiment, the light chain of the antibody against the second antigen comprises the light chain variable region CDR1, CDR2 and / or CDR3, which have been disclosed in the art for anti-PD-L1, or anti-PD-1 or anti-CTLA-4. In one embodiment, the second antigen is PD-L1 and the antibody against the second antigen is an anti-PD-L1 antibody. In certain embodiments, the heavy chain of the anti-PD-L1 antibody comprises the heavy chain variable region V H In certain embodiments, V H comprises the heavy chain variable regions CDR1, CDR2 and CDR3 disclosed for the anti-PD-L1 antibodies. In certain embodiments, the heavy chain comprises the heavy chain variable regions CDR1, CDR2 and CDR3 disclosed for the anti-PD-L1 antibodies. In particular, the heavy chain further comprises a constant region. In certain embodiments, the light chain of the anti-PD-L1 antibody comprises the light chain variable region V L In certain embodiments, V L comprises the light chain variable regions CDR1, CDR2, and CDR3 disclosed for the anti-PD-L1 antibodies. In a specific embodiment, the light chain comprises the light chain variable regions CDR1, CDR2, and CDR3 disclosed for the anti-PD-L1 antibodies. In particular, the heavy chain further comprises a constant region.

[0110] In certain embodiments, the scFv fragment of an anti-4-1BB antibody of the present disclosure may be any of the VFv fragments disclosed for the anti-4-1BB antibody or antibody-binding fragment herein above. H and / or V L In another embodiment, the scFv comprises the heavy chain variable region CDR1, CDR2 and / or CDR3, and / or the light chain variable region CDR1, CDR2 and / or CDR3, which are disclosed for the anti-4-1BB antibody or antibody binding fragment herein above. In particular, the scFv fragment of the anti-4-1BB antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25 or 77, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 26 or 78. In particular, the scFv fragment of the anti-4-1BB antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO:77 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:78. In particular, the scFv fragment of the anti-4-1BB antibody has the V sequences shown in SEQ ID NOs: 1, 2, and 3, respectively. H CDR1, CDR2 and CDR3, and V consisting of the sequences shown in SEQ ID NOs: 4, 5 and 6, respectively L It includes CDR1, CDR2 and CDR3. In particular, V H and V L The linker connecting the two is a flexible linker, such as a linker having glycine and / or serine residues, alone or in combination. In one embodiment, the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, where n is a positive integer of 1 or more, e.g., n is a positive integer of 1 to 7, e.g., n is 2, 3, 4, 5, 6. In one embodiment, n is 1, 2, 3, or 4. In a particular embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:52.

[0111] In certain embodiments, the heavy chain variable region V H teeth, (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 46; or (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 46; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 46, preferably said amino acid modifications do not occur in the CDR regions, more preferably said amino acid modifications occur in the FR regions, such as the FR1, FR2, FR3 or FR4 regions. In certain embodiments, the heavy chain variable regions CDR1, CDR2 and CDR3 disclosed for the anti-PD-L1 antibodies are (i) CDR1, CDR2 and CDR3 derived from a heavy chain variable region comprising or consisting of the sequence set forth in SEQ ID NO: 46; or (ii) CDR1, CDR2 and CDR3 of (i), further comprising a total of from one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of (i). In certain embodiments, the V H CDR1 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 40, or H CDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO:40. In certain embodiments, the V H CDR2 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 41, or H CDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO:41. In certain embodiments, the V H CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 42, or HThe CDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 42. In one embodiment, the three heavy chain variable regions CDR1, CDR2 and CDR3 disclosed for the anti-PD-L1 antibody are: (i) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively; or (ii) CDR1, CDR2 and CDR3 of (i), further comprising a total of from one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of (i). In one embodiment, the heavy chain constant region is or is derived from a human IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region, preferably an IgG1, IgG2 or IgG4 constant region. In another embodiment, the heavy chain constant region comprises: (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 33, 34 or 75; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33, 34 or 75; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence set forth in SEQ ID NO: 33, 34 or 75. In certain embodiments, the light chain variable region V L teeth, (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 47; or (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 47; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 47, preferably said amino acid modifications do not occur in a CDR region, more preferably said amino acid changes occur in a FR region, such as the FR1 region, the FR2 region, the FR3 region or the FR4 region. In certain embodiments, the three heavy chain variable regions CDR1, CDR2, and CDR3 disclosed for the anti-PD-L1 antibodies are: (i) CDR1, CDR2 and CDR3 derived from a light chain variable region comprising or consisting of the sequence set forth in SEQ ID NO: 47; or (ii) CDR1, CDR2 and CDR3 of (i), further comprising a total of from one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of (i). In certain embodiments, the V L CDR1 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 43, or L CDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 43. In certain embodiments, the V L CDR2 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 44, or LCDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO:44. In certain embodiments, the V L CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 45, or L The CDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO:45. In one embodiment, the three light chain variable regions CDR1, CDR2 and CDR3 disclosed for the anti-PD-L1 antibody are: (i) CDR1, CDR2 and CDR3 consisting of the sequences set forth in SEQ ID NOs: 43, 44 and 45, respectively; or (ii) CDR1, CDR2 and CDR3 of (i), further comprising a total of from one to five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three CDRs of (i). In one embodiment, the light chain constant region is or is derived from a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region, preferably a human kappa light chain constant region. In another embodiment, the light chain constant region comprises: (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 35 or 63; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 35 or 63; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence set forth in SEQ ID NO: 35 or 63. In a specific embodiment, the anti-PD-L1 antibody is an antibody disclosed in CN109021107A.

[0112] In one embodiment, the linker in the bispecific antibody of the invention is a flexible linker, such as a linker having glycine and / or serine residues, alone or in combination. In one embodiment, the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, where n is a positive integer equal to or greater than 1, e.g., n is a positive integer from 1 to 7, where n is 2, 3, 4, 5, 6. In one embodiment, n is 1, 2, 3, or 4, preferably 1 or 3. In one embodiment, n is 1, 2, 3, or 4, preferably 1 or 3. In a particular embodiment, the linker of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO: 36, 51 or 52. In a preferred embodiment, the linker of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO: 52, where n=1 or 3.

[0113] In one embodiment, chain 1 of the bispecific antibody is (i) an amino acid sequence comprising or consisting of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 48 or 50; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 48 or 50; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 20, more preferably not more than 10 or 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence set forth in SEQ ID NO: 48 or 50.

[0114] In one embodiment, chain 2 of the bispecific antibody is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 49; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:49; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 20, more preferably not more than 10 or not more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence set forth in SEQ ID NO: 49.

[0115] <Conservative modification>

[0116] In another embodiment, the antibody of the present disclosure comprises heavy and / or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences that differ from those of the anti-4-1BB antibody of the present disclosure by one or more conservative modifications. As is understood in the art, certain conservative sequence modifications can be made while maintaining antigen binding function. See, e.g., Brummell et al. (1993) Biochem 32:1180-8, de Wildt et al. (1997) Prot. Eng. 10:835-41, Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870, Hall et al. (1992) J. Immunol. 149:1605-12, Kelley and O'Connell (1993) Biochem. 32:6862-35, Adib-Conquy et al. (1998) Int. Immunol. 10:341-6, and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0117] Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising a CDR1, CDR2 and CDR3 sequence, and / or a light chain variable region comprising a CDR1, CDR2 and CDR3 sequence, (a) the CDR1 sequence of the heavy chain variable region comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (b) the CDR2 sequence of the heavy chain variable region comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (c) the CDR3 sequence of the heavy chain variable region comprises a sequence listed in Table 1 above, or a conservative modification thereof; and / or (d) the CDR1 and / or CDR2 and / or CDR3 sequences of the light chain variable region comprise the sequences listed in Table 1 above; and / or conservative modifications thereof; and (e) The antibody specifically binds to human 4-1BB.

[0118] In another embodiment, an antibody of the disclosure comprises a heavy and / or light chain variable region sequence that differs from that of an anti-4-1BB antibody of the disclosure by one or more conservative modifications, preferably no modifications occur in the CDRs, and preferably no modifications occur in the FRs.

[0119] In another embodiment, an antibody of the present disclosure comprises a heavy and / or light chain sequence that differs from that of an anti-4-1BB antibody of the present disclosure by one or more conservative modifications, preferably no modifications occur in the CDRs, and preferably no modifications occur in the FRs or in the constant regions.

[0120] In some embodiments, modifications are incorporated into the FRs of the heavy and / or light chain variable regions for the construction of multispecific antibodies, e.g., bispecific antibodies. For example, modifications can be incorporated into the heavy and / or light chain variable regions to form disulfide bonds with each other and improve the stability of scFvs that can be constructed into bispecific antibodies.

[0121] In some embodiments, the modifications are substitutions, additions and / or deletions.

[0122] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the replacement of an amino acid with another amino acid of the same class, such as the replacement of an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. The following table provides examples of substitutions: [ka]

[0123] The antibodies of the present disclosure have one or more of the following functional properties described above, such as specifically binding to human 4-1BB.

[0124] In various embodiments, the antibody can be, for example, a murine, human, humanized or chimeric antibody.

[0125] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or change the binding properties of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In this manner, one or more amino acid residues in the CDR regions of an antibody of the invention can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., the functions described above) using the functional assays described herein.

[0126] <Modified antibodies and modifications>

[0127] The antibody of the present disclosure is the V H / V L An antibody having one or more of the sequences can be prepared using the starting material for engineering a modified antibody. The antibody can have one or both variable regions (i.e., V H and / or V L), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally and alternatively, antibodies can be engineered by modifying residues in the constant region, for example, to alter the effector functions of the antibody.

[0128] In certain embodiments, CDR grafting can be employed to remodel the variable regions of antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular native antibody by constructing expression vectors that contain CDR sequences from that particular native antibody grafted onto framework sequences from a different antibody with different properties (e.g., Riechmann et al. (1998) Nature 332:323-327; Jones et al. (1986) Nature 321:522-525; Queen et al. (1989) Proc. Natl. Acad. USA 86:10029-10033; U.S. Patents 5,225,539, 5,530,101, 5,585,089, 5,693,762, and 6,180,370).

[0129] Thus, another embodiment of the present disclosure relates to isolated monoclonal antibodies or antigen-binding portions thereof comprising a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence comprising a sequence of the present invention as described above, and / or a light chain variable region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence comprising a sequence of the present invention as described above. These antibodies are in the V region of the monoclonal antibodies of the present disclosure. H CDR sequences and V L In addition to comprising CDR sequences, these antibodies may contain different framework sequences.

[0130] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), and in Kabat et al. (1991), cited supra, Tomlinson et al. (1992) J. Mol. Biol. 227:776-798, Cox et al. (1994) Eur. J. Immunol. 24:827-836, the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the heavy chain germline sequence found in the following HCo7 HuMAb mouse can be found in the accompanying Genbank accession numbers 1-69 (NG - 0010109, NT -- 024637&BC070333), 3-33(NG -- 0010109&NT -- 024637) and 3-7 (NG -- 0010109&NT -- As another example, the heavy chain germline sequence found in the following HCo12 HuMAb mouse is available at: - 0010109, NT -- 024637&BC070333), 5-51(NG -- 0010109&NT -- 024637), 4-34(NG -- 0010109&NT -- Available at: 024637), 3-30.3(CAJ556644) & 3-23(AJ406678).

[0131] The antibody protein sequence is compared to compiled protein sequence databases using one of the sequence similarity search methods known to those of skill in the art as Gapped BLAST (Altschul et al. (1997), supra).

[0132] Preferred framework sequences for use in the antibodies of the present invention are structurally similar to the framework sequences used in the antibodies of the present invention. H The CDR1, CDR2 and CDR3 sequences can be grafted into framework regions that have the same sequence as that found in the germline immunoglobulin gene from which the framework sequences are derived. Alternatively, the CDR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequence. For example, it has been found that in certain instances, it is beneficial to mutate residues in the framework regions to maintain and enhance the antigen-binding ability of the antibody (e.g., U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370).

[0133] Other types of variable region modifications include H and / or V L The aim is to improve one or more binding properties (e.g. affinity) of the antibody of interest by mutating amino acid residues in the CDR1, CDR2 and / or CDR3 regions. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be evaluated by in vitro or in vivo assays described herein and provided in the Examples. Preferably, conservative modifications (known in the art) are introduced. The mutations can be amino acid substitutions, insertions or deletions, but are preferably substitutions. Furthermore, typically no more than one, two, three, four and five residues in the CDR regions are altered.

[0134] Thus, in another embodiment, the disclosure provides a method for the preparation of a mAb comprising: (a) a V mAb containing a sequence of the disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; H(b) a CDR1 region comprising an amino acid sequence of the present disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; H (c) a CDR2 region comprising a sequence of the present disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; H (d) a CDR3 region comprising a sequence of the present disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; L (e) a CDR1 region comprising a sequence of the present disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; L and (f) a CDR2 region comprising a sequence of the present disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions. L An isolated anti-4-1BB monoclonal antibody or antigen-binding portion thereof is provided, comprising a heavy chain variable region, including a CDR3 region.

[0135] Engineered antibodies of the invention can be modified, for example, by modifying V H and / or V L Framework modifications include modifications to framework residues in the ribozyme. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived.

[0136] Another type of framework modification involves mutating one or more residues in the framework regions, or one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also called "deimmunization" and is described in detail in U.S. Patent Publication No. 20030153043.

[0137] In addition to or instead of modifications made within the framework or CDR regions, the antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Further, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), modified to alter its glycosylation, or modified to alter one or more functional properties of the antibody.

[0138] In one embodiment, the C is modified such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased). H1 This approach is described in detail in U.S. Pat. No. 5,677,425. H1 The number of cysteine ​​residues in the hinge region of the antibody can be altered to, for example, facilitate assembly of the light and heavy chains and to increase or decrease the stability of the antibody.

[0139] In another embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life. More specifically, the C of the Fc hinge fragment is H2 -C H3 By introducing one or more amino acid mutations in the domain interface region, the antibody has reduced Staphylococcal protein A (SpA) binding compared to native Fc hinge domain SpA binding, an approach that is described in detail in U.S. Patent No. 6,165,745.

[0140] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody is generated (i.e., the antibody lacks glycosylation). Altering the glycosylation can, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more variable region framework glycosylation sites can be eliminated by introducing one or more amino acid substitutions to eliminate glycosylation at that site. Such glycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0141] Additionally and alternatively, antibodies can be made with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues and antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention to produce the antibodies with altered glycosylation. For example, cell lines Ms704, Ms705 and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), and therefore the carbohydrate of the antibodies expressed in the Ms704, Ms705 and Ms709 cell lines lacks fucose. Ms704, Ms705 and Ms709 FUT8 - / -The cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see US Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene (encoding fucosyltransferase). Antibodies expressed in such cell lines exhibit hypofucosylation by reducing and eliminating α-1,6 bond-related enzymes. EP 1,176,195 further describes cell lines with reduced enzymatic activity that adds fucose to N-acetylglucosamine that is either linked to the Fc region of antibodies or does not have enzymatic activity, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO03 / 035835 describes Lec13 cells, a variant CHO cell line that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see Shields et al. (2002) J. Biol. Chem. 277:26733-26740). As described in PCT Publication WO 06 / 089231, antibodies with modified glycosylation profiles can also be produced in chicken eggs. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells such as Lemna. Methods for producing antibodies in plant systems are disclosed in U.S. Patent Application corresponding to Alston & Bird LLP Attorney Docket No. 040989 / 314911, filed August 11, 2006. PCT Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII). Antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, which increases the ADCC activity of the antibodies (see Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibodies can be cleaved off by a fucosidase enzyme.For example, α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).

[0142] Another modification of the antibodies of the present invention is pegylation. By pegylating an antibody, for example, the biological (e.g. serum) half-life of the antibody can be extended. To pegylate an antibody, the antibody and its fragments are typically reacted with polyethylene glycol (PEG), such as reactive ester and aldehyde derivatives of PEG, under conditions where one or more PEG groups are attached to the antibody and antibody fragment. Preferably, pegylation is carried out via acylation and alkylation reactions with reactive PEG molecules (and similar reactive water-soluble polymers). The term "polyethylene glycol" as used herein is intended to encompass any form of PEG used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol and polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EPO154316 and EP0401384.

[0143] <Physical properties of antibodies>

[0144] The antibodies of the present invention may be characterized by their various physical properties in order to detect and / or distinguish their different classes.

[0145] For example, an antibody may contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody and alteration of the antibody's pK due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some instances, it may be preferable to have an anti-PD-1 antibody that does not contain variable region glycosylation, which can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0146] In a preferred embodiment, the antibody does not contain any asparagine isomerism sites. Deamidation of asparagine occurs at NG and DG sequences and can result in the generation of isoaspartic acid residues that introduce kinks into the polypeptide chain and reduce its stability (the isoaspartic acid effect).

[0147] Each antibody has a unique isoelectric point (pI), which generally lies in the pH range of 6-9.5. The pI of an IgG1 antibody is typically in the pH range of 7-9.5, and the pI of an IgG4 antibody is typically in the pH range of 6-8. It has been speculated that antibodies with pIs outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-4-1BB antibody that contains a pI value that is in the normal range. This can be achieved by selecting an antibody with a pI in the normal range or by mutating charged surface residues.

[0148] <Nucleic acid molecules encoding the antibodies of the present invention>

[0149] In another aspect, the invention provides nucleic acid molecules encoding the heavy and / or light chain variable regions or CDRs of the antibody of the invention. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components and other contaminants, such as other cellular nucleic acids and proteins, by standard techniques. The nucleic acids of the invention can be, for example, DNA and RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0150] The nucleic acids of the present invention can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), cDNAs encoding the light and heavy chains of the antibodies made by the hybridomas can be obtained by standard PCR amplification and cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding such antibodies can be retrieved from the gene library.

[0151] A preferred nucleic acid molecule of the present invention is the V H Array and V L Preferred nucleic acid molecules of the invention include nucleic acid molecules encoding the V and CDRs of a multispecific antibody (e.g., a bispecific antibody). H Array and V L The present invention further includes nucleic acid molecules encoding the sequence or each strand thereof. H and V L Once the DNA fragments encoding the segments are obtained, they can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes, or multispecific (bispecific) antibody chain genes. Lor V H The DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0152] In one embodiment, exemplary nucleic acids of the invention include nucleic acids encoding an amino acid sequence selected from any of SEQ ID NOs: 25-32, 48, 50, 77, and 78, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any of SEQ ID NOs: 25-32, 48, 50, 77, and 78.

[0153] In another embodiment, a nucleic acid of the invention comprises a nucleotide sequence selected from any of SEQ ID NOs: 65, 66, 73, 74, 79 or 80, or a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence selected from any of SEQ ID NOs: 65, 66, 73, 74, 79 or 80.

[0154] V H The isolated DNA encoding the V H The DNA encoding the heavy chain constant region (C H1 , C H2 and C H3The heavy chain constant region may be converted to a full-length heavy chain gene by operably linking to another DNA molecule encoding the heavy chain constant region (V), which is a nucleotide sequence ...). The sequences of human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably an IgG1, IgG2 or IgG4 constant region, or a constant region having a mutation, for example a constant region comprising or consisting of the sequence shown in SEQ ID NO: 33, 34 or 75, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. H The coding DNA is heavy chain C H1 It can be operably linked to another DNA molecule encoding only the constant region.

[0155] V L The isolated DNA encoding the V L The coding DNA for the light chain constant region C L The light chain constant region can be converted to a full-length light chain gene (and a Fab light chain gene) by operably linking it to another DNA molecule encoding the light chain constant region. The sequences of human light chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region, e.g., a light chain constant region comprising or consisting of a sequence set forth in SEQ ID NO: 35 or 63, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0156] To generate the scFv gene, H and V L The coding DNA fragment is operably linked to a flexible linker, e.g., another fragment encoding the amino acid sequence (Gly4-Ser)3, thereby forming a V H and V L The sequence is connected to the V L and VH The regions can be expressed as a contiguous single-chain protein (e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0157] Preparation of the Antibodies of the Disclosure

[0158] The invention provides a process for preparing an antibody of the invention, said process comprising the steps of culturing a host cell of the invention under conditions allowing expression of the antibody, and recovering the antibody construct obtained from the culture.

[0159] The monoclonal antibodies (mAbs) of the present invention can be prepared using the well-known somatic cell hybridization technique (Kohler and Milstein (1975) Nature 256:495). Other embodiments for preparing monoclonal antibodies include viral and oncogenic transformation of B lymphocytes and phage display techniques. Chimeric and humanized antibodies are also well known in the art. See, e.g., U.S. Patent Nos. 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762, and 6,180,370, the contents of which are incorporated herein by reference in their entireties.

[0160] The bispecific antibody of the present disclosure can be constructed according to the bispecific antibody format using a known platform for bispecific antibodies.There are several methods known in the art for producing bispecific antibodies, such as those described in U.S. Patent No. 4,816,567 and U.S. Publication No. 2013 / 0078249, which are incorporated herein by reference in their entirety.

[0161] <Preparation of transfectomas producing the monoclonal antibodies of the present invention>

[0162] Antibodies of the invention can be produced, for example, in host cell transfectomas by a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial and full-length light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. As used herein, the term "operably linked" means that the antibody genes are ligated into a vector such that the transcriptional and translational regulatory sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0163] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription and translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), Simian Virus 40 (SV40), and adenovirus (e.g., the adenovirus major late promoter (AdMLP) and polyoma). Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter and the β-globin promoter. In addition, regulatory elements can be composed of sequences from different sources, for example, the SRα promoter system contains sequences derived from the SV40 early promoter and the long terminal repeat of the human T-cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0164] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In a preferred embodiment, the variable regions are used to express the V H The segment is C in the vector H Functionally linked to the segment, V L The segment is C in the vector L Full-length antibody genes of any antibody isotype are generated by inserting the variable regions into an expression vector already encoding the heavy and light chain constant regions of the isotype of interest such that they are operably linked to the segments. Additionally and alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0165] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the Neo gene (for G418 selection).

[0166] For expression of the light and heavy chains, the heavy and light chain encoding expression vectors are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic and eukaryotic host cells (e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc.). Although it is theoretically possible to express the antibodies of the present invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred, since such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded antibodies with immunological activity.

[0167] A preferred mammalian host cell for expressing a recombinant antibody of the invention is Chinese hamster ovary (CHO) cells (DHFR, used with the DHFR selection marker (e.g., R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621)). - Cells that are suitable for use with the present invention include CHO cells (including Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), NSO myeloma cells, COS cells, and SP2. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene has been introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or, preferably, secretion of the antibody into the medium in which the host cell is grown. The antibody can be recovered from the medium by standard protein purification methods.

[0168] <Pharmaceutical Composition>

[0169] In another aspect, the disclosure provides a pharmaceutical composition comprising one or more antibodies (or antigen-binding portions thereof, or bispecific molecules) of the disclosure formulated with a pharma- ceutically acceptable carrier. The antibodies (or antigen-binding portions thereof, or bispecific molecules) may be administered individually if the composition comprises multiple antibodies (or antigen-binding portions thereof, or bispecific molecules). The compositions may optionally contain one or more additional pharma- ceutical active ingredients, such as therapeutic agents, such as anti-cancer, anti-bacterial, or anti-asthmatic drugs, other antibodies or drugs, e.g., chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against immune checkpoint molecules such as PD-1), anti-infective agents, small molecule agents, or immunomodulatory agents.

[0170] As used herein, the term "therapeutic agent" encompasses substances effective in the prevention or treatment of neoplasia (e.g., cancer), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against immune checkpoint molecules), anti-infective agents, immunomodulatory agents, or small molecule drugs.

[0171] Pharmaceutical compositions may contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners and emulsifiers, solid binders, dispersion and suspension aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is described in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0172] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal and epidermal administration (e.g., by injection and infusion). Depending on the route of administration, the active compound may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. The term "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection and infusion. Alternatively, the antibody of the present invention may be administered via non-parenteral routes, such as topical, epidermal and mucosal routes of administration, e.g., intranasally, orally, intravaginally, rectally, sublingually and topically.

[0173] The pharmaceutical compositions may be in the form of sterile aqueous solutions and dispersions. They can also be formulated into microemulsions, liposomes, and other ordered structures suitable to high drug concentration.

[0174] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular route of administration, and will generally be that amount of the composition that produces a therapeutic effect. In general, the amount of active ingredient, based on 100% of the total of active ingredient and pharma- ceutically acceptable carrier, will be from about 0.01% to about 99%, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30%.

[0175] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, it may be administered in a single bolus, in divided doses over time, or the dose may be proportionally reduced and increased depending on the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form in terms of ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dose for the subject to be treated. Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained release formulation, in which case the frequency of administration may need to be reduced.

[0176] For administration of antibodies, dosages range from about 0.0001-100 mg / kg, more typically 0.01-5 mg / kg of host body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, or 1-10 mg / kg body weight. Exemplary treatment regimes involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, and once every three to six months. Preferred dosing regimes for the anti-4-1BB antibodies of the invention include intravenous administration at 1 mg / kg body weight and 3 mg / kg body weight, with each agent given simultaneously by one of the following dosing regimes: (i) once every four weeks for six doses, followed by once every three months, (ii) once every three weeks, and (iii) once at 3 mg / kg body weight followed by once every three weeks at 1 mg / kg body weight. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods about 25-300 μg / ml.

[0177] A "therapeutically effective dose" of the anti-4-1BB antibody of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of a disorder or disorder due to the affliction of a disease. For example, for the treatment of a subject with a tumor, a "therapeutically effective dose" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least 60%, and even more preferably at least about 80% compared to an untreated subject. A therapeutically effective amount of a therapeutic compound can reduce tumor size or ameliorate symptoms in a subject. The subject can typically be a human or other mammal.

[0178] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, colLAGen, polyorthoesters, polylactic acid, etc. For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0179] Therapeutic compositions can be administered by medical devices such as: (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556), (2) microinfusion pumps (U.S. Pat. No. 4,487,603), (3) transdermal devices (U.S. Pat. No. 4,486,194), (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224), and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0180] In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo, for example, to ensure that the therapeutic antibodies of the invention cross the blood-brain barrier, they can be formulated into liposomes and may further include targeting moieties that facilitate selective delivery to specific cells and organs. For example, U.S. Pat. al. (1995) FEBS Lett. 357:140, M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180, Briscoe et al. (1995) Am. J. Physiol. 1233: 134, Schreier et al. (1994) J. Biol. Laukkanen (1994) FEBS Lett. 346:123, as well as Killion and Fidler (1994) Immunomethods 4:273.

[0181] Combination products

[0182] In another aspect, the disclosure provides combination products comprising one or more antibodies (or antigen-binding portions thereof, or bispecific molecules) of this disclosure and one or more additional therapeutic agents, such as other antibodies or drugs, such as anti-cancer drugs, anti-bacterial drugs, or anti-asthma drugs, e.g., chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against immune checkpoint molecules such as PD-1), anti-infective agents, small molecule drugs, or immunomodulatory agents.

[0183] A "combination" is a fixed or non-fixed combination or kit of parts in dosage unit form for combined administration, in which two or more therapeutic agents can be administered alone at the same time or separately within a time interval, particularly when the combination partners can exhibit a collaboration, e.g., synergistic effect, within these time intervals. The term "fixed combination" means that the antibody of the present invention and the combination partner (e.g., other therapeutic agents, such as immunosuppressants or immunomodulators, such as anti-inflammatory agents) are administered to the patient at the same time in the form of a single entity or dose. The term "non-fixed combination" means that the antibody of the present invention and the combination partner (e.g., other therapeutic agents, such as immunosuppressants or immunomodulators, such as anti-inflammatory agents) are administered to the patient simultaneously, together, or sequentially as separate entities, and there is no specific time limit, provided that such administration provides the patient with therapeutically effective levels of the two compounds. The latter also applies to cocktail therapy, such as the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

[0184] Uses and Methods

[0185] Compositions comprising the antibodies or antigen-binding portions thereof, or bispecific molecules of the invention have many in vitro and in vivo utilities, including, for example, the treatment of cancer, infectious diseases, and autoimmune diseases. The antibodies can be administered to human subjects, for example, in vivo, to alleviate these diseases.

[0186] In one aspect, the invention provides a method of modulating an immune response in a subject. In another aspect, the present invention provides a method for activating T cells or inducing T cell-mediated anti-tumor activity. In one embodiment, T cells are CD8+ T cells. In another aspect, the present invention provides a method for reducing Treg cells, for example CD4+ T cells. In one embodiment, activating a T cell includes stimulating T cell cytokine secretion, for example, T cell secretion of IL-12. In one embodiment, the invention provides a method of activating the 4-1BB signaling pathway.

[0187] In some embodiments, the methods include administering an antibody or antigen-binding fragment, a pharmaceutical composition or formulation, a combination product, or a nucleic acid of the invention.

[0188] In one embodiment, the antibody of the invention is a bispecific antibody that binds to 4-1BB and PD-L1, as described above. In one embodiment, the invention provides a method of treating cancer in a subject comprising administering a bispecific antibody that binds 4-1BB and PD-L1 of the present disclosure, or a pharmaceutical composition or combination comprising same.

[0189] In some embodiments, the cancer is tumor immune evasion.

[0190] In some embodiments, cancer includes solid and non-solid cancers and metastatic lesions. In one embodiment, solid cancer includes malignant tumors. In one embodiment, solid cancer includes malignant tumors. Cancer can be early stage, intermediate stage, or advanced or metastatic cancer. In some embodiments, cancer is a cancer that requires T cell activation, such as cancer with T cell dysfunction.

[0191] In some embodiments, cancers display increased levels of PD-L1 protein expression or increased levels of nucleic acid encoding PD-L1, e.g., compared to levels in normal subjects or normal cells.

[0192] Preferably, the cancer is a cancer of the digestive tract (gastrointestinal tract), such as colorectal cancer (large intestine cancer), rectal cancer or colon cancer.

[0193] In some embodiments, the benefits of treating cancer include: (i) Inhibiting PD-L1 nucleic acid or protein levels. (ii) Blocking the binding of PD-L1 to receptors such as PD-1. (iii) Activating the 4-1BB signaling pathway. (iv) T cell activation, e.g., increasing the proliferation of CD8+ T cells or decreasing Treg cells, e.g., CD4+ T cells. (v) Any combination of one or more of the above.

[0194] In another aspect, the present disclosure provides a method of combination therapy in which an anti-4-1BB antibody or antigen-binding portion thereof, or a bispecific molecule of the present disclosure is administered in combination with one or more therapeutic agents, such as an additional antibody, effective to alleviate cancer, an infectious disease, or an autoimmune disease in a subject. In one embodiment, the present disclosure provides a method of treating a cancer disease in a subject, comprising administering to the subject an anti-4-1BB antibody (or antigen-binding portion thereof, or a bispecific antibody) and one or more therapeutic agents, such as an additional antibody, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In certain embodiments, the subject is a human.

[0195] Activation of 4-1BB signaling can further be combined with standard disease treatments, for example, activation of 4-1BB signaling can be combined with administration of the above-mentioned antibodies or chemical anticancer agents.

[0196] Thus, combination therapy includes the administration of an anti-4-1BB antibody or antigen-binding portion thereof, or bispecific molecule of the present disclosure in combination with pharmaceutical compositions and combinations thereof, as well as other therapies, such as therapeutic modalities and / or other therapeutic agents, preferably therapeutic modalities including surgery and / or radiation therapy, and / or other therapeutic agents selected from the group consisting of chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule agents, or immunomodulatory agents.

[0197] The combinations of therapeutic agents discussed herein may be administered simultaneously as a single composition in a pharma- ceutically acceptable carrier or as separate compositions with each agent in a pharma- ceutically acceptable carrier. In another embodiment, the combinations of therapeutic agents may be administered sequentially.

[0198] Additionally, when multiple administrations of the combination therapy are administered sequentially, the order of sequential administration can be reversed or kept the same at each administration time point, and sequential administration can be combined with simultaneous administration, or any combination thereof. EXAMPLES

[0199] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, GenBank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. Working Example Example 1: Phage panning, screening and affinity maturation

[0200] Phage Library

[0201] An antibody single chain phage display library was created by cloning a repertoire of light chain variable regions (VL) and a repertoire of heavy chain variable regions (VH). The heavy and light chain repertoires were created by PCR amplification from human lymphocytes mainly taken from peripheral blood. The VL and VH repertoires were mixed and subjected to PCR using overlapping primers. The final format of the antibody was a single chain Fv (scFv) with VH and VL fragments linked with a flexible linker peptide (GGGGSGGGGSGGGGS (SEQ ID NO: 36)).

[0202] Phage library panning against human 4-1BB

[0203] Selection of phage particles displaying specific scFv fragments was performed using Immuno 96 MicroWell TM The plate was coated with 50 μg / mL of 4-1BB recombinant protein (Sinobiological, Cat. No. 10041-H08H) in phosphate-buffered saline (PBS) overnight at 4°C. After blocking with 2% (w / v) milk powder in PBS (2% MPBS), the plate was incubated for approximately 10 min. 11 A library containing 10 phage particles was added and the plate was incubated for 2 h at room temperature (RT, 25-28 °C). Unbound phages were removed by washing the plate 10-20 times with PBS containing 0.1% Tween 20 (PBST) followed by 10-20 washings with PBS. Bound phages were eluted by incubation with 50 μl of 1 μg / μl trypsin for 10 min, followed by incubation with 50 μl of 50 mM glycine-HCl, pH 2.0 for 10 min and immediate neutralization with 50 μl of 200 mM Na2HPO4, pH 7.5. Four rounds of panning were performed.

[0204] Phage screening

[0205] From the third and fourth rounds of panning, phages were picked up and tested for human 4-1BB binding. Specifically, human 4-1BB (Sinobiological, Cat. No. 10041-H08H) was coated onto a 96-well plate at 0.1 μg / mL, and single clone phage phage was added to the plate. Unbound phage was then washed away, and bound phage was detected by anti-M13 secondary antibody (Abcam, Cat. No. ab50370).

[0206] The ELISA positive clones were sequenced, from which 28 unique sequences were identified, including clones 41BB-2, 41BB-9, 41BB-13, and 41BB-27. The amino acid sequence ID numbers of the heavy / light chain variable regions of exemplary anti-4-1BB antibodies and their CDRs (as defined by Kabat numbering) are shown in Table 2. These heavy / light chain variable region sequences were used to prepare full antibodies 41BB-2, 41BB-9, 41BB-13, and 41BB-27 with human IgG2 (SEQ ID NO: 33) or IgG1 (SEQ ID NO: 34) or IgG4 heavy chain constant region (SEQ ID NO: 75) and human light chain lambda constant region (SEQ ID NO: 35), in which the C-terminus of the heavy chain variable region was linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region was linked to the N-terminus of the light chain constant region. Additionally, the light chain variable region of antibody 41BB-2 included the amino acid sequence of SEQ ID NO: 26, X1=S.

[0207] In addition, the light chain variable region of the 41BB-2 antibody has a mutation from S to G at the final site and is connected to an IgG1 heavy chain constant region (SEQ ID NO: 34). The resulting denatured 41BB-2 could be prepared with higher purity than 41BB-2 having an IgG1 heavy chain constant region (data not shown). [Table 2]

[0208] The antibody having the above sequence is prepared and purified by a general method known in the art. Specifically, the nucleotide sequences encoding the heavy and light chains of the antibody were inserted into the expression vector pcDNA3.1 (Invitrogen). The vector was expressed as ExpiCHO TM CHO-S cells were co-transfected using the ExpiCHO expression system (ThermoFisher) according to the manufacturer's instructions. TM After culturing in expression medium for 12 days, the culture supernatant was collected and sent for purification by protein A affinity chromatography (GE healthcare) according to the manufacturer's instructions. Example 2: Binding of Exemplary Anti-4-1BB Antibodies to Human 4-1BB

[0209] An ELISA assay was performed to determine the relative binding capabilities of the antibodies to human 4-1BB.

[0210] Human 4-1BB (Sinobiological, Catalog No. 10041-H08H) in carbonate buffer pH 9.6 was immobilized on a 96-well plate by overnight incubation at 4°C at 25ng / well. The plate was then blocked by incubating with 1% BSA in PBS at 37°C for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween20). Serially diluted anti-4-1BB antibodies and urelumab control (Urelumab control: prepared according to WO2005035584 using heavy and light chain amino acid sequences shown in SEQ ID NOs: 37 and 38), respectively, were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the immobilized protein in the plate for 1 hour at 37°C. After binding, plates were washed three times with PBST and incubated with peroxidase-labeled goat anti-human IgG F(ab')2 antibody (Jackson Immuno Research, Cat. No. 109-035-097) diluted 1 / 20000 in binding buffer for 1 hour at 37°C, washed again, developed with TMB (ThermoFisher, Cat. No. 34028) for 15 minutes, and then stopped with 1M H2SO4. Each plate well contained 50 μL of solution at each step.

[0211] The absorbance was measured at 50 nm to 620 nm. EC for antibodies binding to human 4-1BB 50 The values ​​and binding curves are shown in Figures 1A to 1D, which suggest that the anti-4-1BB antibodies 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2, and 41BB-27-IgG2 specifically bound to human 4-1BB with higher binding capacity than the urelumab control. Example 3: Exemplary anti-4-1BB antibodies cross-react with Rhesus 4-1BB but not with human CD40, human HVEM, human OX40, human CD27, or human GITR

[0212] ELISA assays were performed to determine the binding activity of exemplary anti-4-1BB antibodies to recombinant human CD40, human HVEM, human OX40, human CD27, human GITR or Rhesus 4-1BB.

[0213] Human CD40 (Sinobiological, Catalog No. 10774-H08H), human HVEM (Sinobiological, Catalog No. 10334-H03H), human OX40 (Sinobiological, Catalog No. 10481-H08H), human CD27 (Sinobiological, Catalog No. 10039-H31H), human GITR (Sinobiological, Catalog No. 13643-H08H) and Rhesus 4-1BB (Sinobiological, Catalog No. 90847-K08H) were immobilized on 96-well plates by overnight incubation at 4°C, 25ng / well, respectively, in carbonate buffer pH 9.6. The plates were then blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. The plates were then washed three times with PBST (PBS containing 0.05% Tween 20). Anti-4-1BB antibody and urelumab were prepared at 500ng / mL each in binding buffer (PBS with 0.05% Tween20 and 0.5% BSA) and incubated with immobilized proteins for 1 hour at 37°C. After binding, plates were washed 3 times with PBST and incubated with peroxidase-labeled anti-human IgGF(ab')2 antibody (Jackson Immuno Research, Cat. No. 109-035-097) diluted 1 / 20000 in binding buffer for 1 hour at 37°C, washed again, and developed with TMB (ThermoFisher Cat. No. 34028) for 15 minutes and then stopped with 1M H2SO4. Each plate well contained 50μL of solution at each step.

[0214] The absorbance was measured at 450 nm to 620 nm and is shown in Figures 2A-2B. It can be seen that the anti-4-1BB antibodies 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2, and 41BB-27-IgG2 did not bind to human CD40, human HVEM, human OX40, human CDGITR, or human GITR, but cross-reacted with Rhesus 4-1BB. Urelumab did not bind to any of these proteins. Example 4: Binding of exemplary anti-4-1BB antibodies to cell surface 4-1BB and induction of NFκB reporter gene luminescence signal

[0215] The agonistic effect of exemplary anti-4-1BB antibodies on human 4-1BB signaling was assessed in a HEK293-NFκB-Luc-human 4-1BB reporter gene assay.

[0216] In Example 1, whole antibodies 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2, 41BB-27-IgG2, 41BB-9-IgG1, 41BB-13-IgG1, 41BB-27-IgG4, 41BB-2-IgG1 were prepared and purified.

[0217] All antibodies, 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2, and 41BB-27-IgG2, were evaluated as follows.

[0218] Briefly, HEK293 cell line (ATCC, Catalog No. CRL-1573) was maintained in DMEM medium containing 10% FBS in a humidified incubator at 37° C. and 5% CO2. Nucleic acid sequences encoding human 4-1BB (amino acid of NP_001552.2 shown in SEQ ID NO: 39) and pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, Catalog No. E849A) were co-transfected into HEK293 cells using Lipofectamine TM2000 transfection reagent (Invitrogen, Catalog No. 11668019), and clones stably expressing human 4-1BB and NF-κB-Luc were obtained by limiting dilution. The HEK293-NFκB-Luc-4-1BB cells obtained in DMEM medium containing 10% FBS were seeded on 96-well plate (50000 cells / well) on the first day and cultured overnight in CO2 incubator. On the second day, the medium on the 96-well plate was discarded, and then anti-4-1BB antibody or urelumab was serially diluted in DMEM containing 1% FBS, whether pretreated with 2x concentration of goat anti-human IgG (Jackson Immuno Research, Catalog No. 109-005-098), added to the plate, and co-cultured with HEK293-NFκB-Luc-4-1BB cells. The plate was incubated in an incubator at 37°C and 5% CO2 for 6 hours. Then, 60 μL of One-Glo™ reagent (Promega, Cat. No. E6130) was added to the wells of the assay plate and luminescence was measured using a luminescence plate reader (Tecan F200 Pro). 50 Values ​​were determined, and representative curves for the anti-4-1BB antibody are shown in Figures 3A to 3G.

[0219] Goat anti-human IgG reacted with the Fc portion of anti-4-1BB antibodies to cross-link two or more anti-4-1BB antibodies. The formation of antibody dimers or polymers may contribute to better agonistic effects on 4-1BB signaling.

[0220] The results showed that 41BB-2-IgG2, 41BB-9-IgG2, 41BB-13-IgG2 and 41BB-27-IgG2 all bind to 4-1BB expressed on the cell surface, and accordingly, whether they are cross-linked or not, can activate 4-1BB signaling and induce luminescent expression of NFκB reporter gene. However, without cross-linking, the 4-1BB antibody of the present invention has very low agonistic effect on human 4-1BB signaling, but with cross-linking, it has higher agonistic effect. The agonistic effect of the four 4-1BB clones is dependent on Fc cross-linking.

[0221] All antibodies 41BB-9-IgG1, 41BB-13-IgG1, 41BB-27-IgG4, 41BB-2-IgG1 were tested as follows.

[0222] Briefly, HEK293 cell line (ATCC, Catalog No. CRL-1573) was maintained in DMEM medium containing 10% FBS in a humidified incubator at 37° C. and 5% CO2. The nucleic acid sequence encoding human 4-1BB (amino acid of NP_001552.2 shown in SEQ ID NO: 39) in pIRESpuro3 plasmid and pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, Catalog No. E849A) was co-transfected into HEK293 cells using Lipofectamine TM2000 transfection reagent (Invitrogen, Catalog No. 11668019), and clones stably expressing human 4-1BB and NF-κB-Luc were obtained by limiting dilution after screening with puromycin (Gibco, Catalog No. A1113802) and hygromycin. The HEK293-NFκB-Luc-human 4-1BB cells obtained in DMEM medium containing 10% FBS were seeded in 384-well plate (20000 cells / well in 40μL).Then, 10μL / well of anti-4-1BB antibodies 41BB-9-IgG1, 41BB-13-IgG1, 41BB-27-IgG4, 41BB-2-IgG1, or urelumab (starting from 50μg / mL in 3-fold dilution series) were serially diluted in DMEM containing 10% FBS, whether or not they were pretreated with goat anti-human IgG (Jackson Immuno Research, Catalog No. 109-005-098) diluted in 2-fold dilution series, and added to the plate.The plate was incubated in an incubator at 37℃ and 5% CO2 for 6 hours. After incubation, 30 μL of One-Glo™ Reagent (Promega, Cat. No. E6130) was added to the wells of the assay plate and luminescence was measured using a luminescence plate reader (Tecan F200 Pro). The results are shown in Figures 4A and 4B.

[0223] The goat anti-human IgG reacted with the Fc portion of the anti-4-1BB antibodies to cross-link two or more anti-4-1BB antibodies.

[0224] The results showed that with cross-linking agents, 41BB-2-IgG1, 41BB-9-IgG1, and 41BB-13-IgG1, except for 41BB-27-IgG4, activated human 4-1BB signaling and induced much higher NFκB reporter gene signals than urelumab. On the other hand, without cross-linking agents, the four 4-1BB antibodies induced much lower NFκB reporter gene signals than urelumab. The agonistic effects of the four 4-1BB clones are dependent on Fc cross-linking. Example 5: Expression and purification of bispecific antibodies

[0225] Two bispecific antibodies, P4B-2 and P4B-3, were constructed and their structures are shown in Figure 5B (P4B-2) and Figure 5A (P4B-3). Specifically, the bispecific P4B-2 was constructed as follows, by linking two scFvs of the 4-1BB antibody to the N-terminus of the two heavy chains of the whole PD-L1 antibody, respectively: V from 4-1BB antibody H (Anti-4-1BB V H )-Linker-4-1BB antibody V L (Anti-4-1BB V L )-Linker from PD-L1 antibody-V H (Anti-PD-L1 V H )-heavy chain constant region, A heavy chain consisting of the N-terminus to the C-terminus; The light chain from the PD-L1 antibody (the light chain of anti-PDL1, i.e., the V L ) and a bispecific antibody comprising: The bispecific P4B-3 was constructed as follows, linking the two scFvs of the 4-1BB antibody to the C-terminus of the two heavy chains of the whole PD-L1 antibody, respectively: V from PD-L1 antibody H (Anti-PD-L1 V H )-heavy chain constant region-linker-V from 4-1BB antibody H (anti-4-1BB V H )-Linker-4-1BB antibody V L (Anti-4-1BB V L), and a heavy chain consisting of, from the N-terminus to the C-terminus: The light chain from the PD-L1 antibody (the light chain of anti-PDL1, i.e., the V L ) and a bispecific antibody comprising:

[0226] Nucleotides encoding bispecific antibodies and controls were generated by gene synthesis (Genscript) and cloned into the expression vector pcDNA3.1 (Invitrogen). The nucleic acid encoding the light chain variable region was inserted into the expression vector pcDNA3.1 (Invitrogen) containing the nucleic acid encoding the light chain constant region to construct a vector expressing the light chain of the antibody, and the nucleic acid encoding the heavy chain variable region was inserted into the expression vector pcDNA3.1 (Invitrogen) containing the nucleic acid encoding the heavy chain constant region to construct a vector expressing the heavy chain of the antibody. The resulting vectors were co-transfected into CHO-S cells at a molar ratio of 1:1 using the ExpiCHOTM Expression System (ThermoFisher, Cat. No. A29133) according to the manufacturer's instructions. The transfected cells were cultured in ExpiCHOTM Expression Medium for 12 days, after which the culture supernatant was collected and purified by Protein A affinity chromatography (GE Healthcare) according to the manufacturer's instructions.

[0227] The amino acid and nucleic acid sequences of the different regions are shown in Table 3. [Table 3] Example 6: Antibodies that bind to human PD-L1 and 4-1BB proteins

[0228] Human PD-L1, His-tagged protein (ACRO, Cat. No. PD1-H5229) was immobilized on a 96-well plate (Hangzhou Xinyou, Cat. No. 100096H) by incubating in carbonate buffer (pH 9.6) overnight at 4° C. The plate was then blocked by incubating with 1% BSA in PBS at 37° C. for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of P4B-2, P4B-3 and negative control IgG (heavy chain: SEQ ID NO: 82; light chain: SEQ ID NO: 83) from 2 nM to 0.003 nM were prepared in dilution buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with the immobilized proteins above at 37 °C for 1 h, after which the plate was washed three times with PBST and incubated with Peroxidase Affinipure Goat Anti-Human IgG, Fcγ Fragment (Jackson ImmunoResearch, Cat. No. 109-035-098) specifically diluted 1 / 20000 in dilution buffer at 37 °C for 1 h, then washed again with PBST. TMB (Thermo, Cat. No. 34028) was added to the plate to initiate the reaction. After 15 min, the reaction was stopped with 1 M H2SO4. The absorbance at 450 nm to 620 nm was measured. EC 50 and representative binding curves are shown in FIG.

[0229] The results showed that both antibodies, P4B-2 and P4B-3, had EC values ​​of 0.02395 nM and 0.02887 nM, respectively. 50 They demonstrated that it can bind to the human PD-L1 protein.

[0230] Human 4-1BB, human Fc tag protein (SEQ ID NO: 81, Hu4-1BB-hFc) was immobilized on a 96-well plate (Hangzhou Xinyou, Catalog No. 100096H) by incubating overnight at 4°C in carbonate buffer (pH 9.6). The plate was then blocked by incubating with 1% BSA in PBS at 37°C for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween20). Serial dilutions of P4B-2, P4B-3 and negative control IgG (heavy chain: SEQ ID NO: 82; light chain: SEQ ID NO: 83) from 6.65 nM to 0.009 nM were prepared in dilution buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the immobilized protein Hu4-1BB-hFc at 37°C for 1 hour. Plates were then washed three times with PBST and incubated with Peroxidase Affinipure Goat Anti-Human IgG, F(ab')2 Fragment (Jackson ImmunoResearch, Cat. No. 109-035-097) specifically diluted 1 / 10000 in dilution buffer for 1 hour at 37°C, then washed again with PBST. TMB (Thermo, Cat. No. 34028) was added to the plate to initiate the reaction. After 10 minutes, the reaction was stopped with 1M H2SO4. Absorbance was measured at 450nm-620nm. EC of clones for binding to human 4-1BB 50 and representative binding curves are shown in FIG.

[0231] The results showed that both antibodies, P4B-2 and P4B-3, had EC values ​​of 0.1126 nM and 0.07234 nM, respectively. 50 We demonstrated that it can bind to human 4-1BB protein.

[0232] Thus, the two bispecific antibodies P4B2 and P4B3 inhibited EC 50 It has low affinity for human PD-L1 and can bind to both human PD-L1 and human 4-1BB proteins. Example 7: Binding Affinity Binding affinity to PD-L1 protein

[0233] The kinetic binding activity of P4B-3 antibody to human PD-L1 protein (ACRO, catalog no. PD1-H5229) and cynomolgus monkey PD-L1 protein (Sino Biological, catalog no. 90251-C08H) was measured by ForteBio Octet RED96 (Fortebio).

[0234] P4B-3 antibody was coated onto a pre-equilibrated anti-human IgG Fc capture (AHC) biosensor (Fortebio, Cat. No. 18-5060). Human PD-L1 (ACRO, Cat. No. PD1-H5229) and cynomolgus PD-L1 (Sino Biological, Cat. No. 90251-C08H) proteins were used as analytes and captured by the antibody. Data sets were fitted to a 1:1 binding model using Octet software.

[0235] Table 4 summarizes the affinity of the P4B-3 bispecific antibody of the invention to human and cynomolgus PD-L1 proteins. [Table 4]

[0236] The results showed that antibody P4B-3 had K values ​​of 0.29 nM and 0.061 nM, respectively. D These results suggest that the antibody specifically binds to both human PD-L1 and cynomolgus monkey PD-L1. Binding affinity to 4-1BB protein

[0237] The kinetic binding activity of P4B-3 antibody against human 4-1BB protein (Sino Biological, Cat. No. 10041-H08H) was measured by ForteBio Octet RED96 (Fortebio).

[0238] Anti-4-1BB antibody was coated onto a pre-equilibrated anti-human IgG Fc capture (AHC) biosensor (Fortebio, Cat. No. 18-5060). Human 4-1BB (Sino Biological, Cat. No. 10041-H08H) was used as the analyte and captured by the antibody. Data sets were fitted to a 1:1 binding model using Octet software.

[0239] Table 5 summarizes the affinities of anti-human 4-1BB antibodies to the human 4-1BB protein. [Table 5]

[0240] The results showed that antibody P4B-3 had an EC 50 These results suggest that the antibody can bind to human 4-1BB protein. Example 8: 4-1BB reporter gene assay of P4B-3 in the HEK293-4-1BB / NFκB system

[0241] The activation of P4B-3 bispecific antibody was evaluated with a PD-L1-dependent 4-1BB reporter gene system. HEK293-NFκB-Luc-human 4-1BB cell line, A375-PD-L1 target cell line and CHO-K1-PDL-1 cell line were developed by Leadsbiolabs.

[0242] The HEK293-NFκB-Luc-human 4-1BB cell line is obtained according to the procedure described in paragraph

[0222] , Example 4.

[0243] A375 (Cell Bank, Type Culture Collection, Chinese Academy of Sciences, Catalog No. SCSP-533) parental cell line was transfected with human PD-L1 gene (SEQ ID NO:62; encoding the amino acid sequence of SEQ ID NO:61) using LipofectamineTM2000 transfection reagent (Invitrogen, Catalog No. 11668019). A stable single cell clone highly expressing human PD-L1 was selected after screening with puromycin (Gibco, Catalog No. A1113802) to obtain A375-PD-L1 cells.

[0244] CHO-K1 (ATCC Cat. No. CCL-61) parental cell line was transfected with human PD-L1 gene (SEQ ID NO:62: encoding the amino acid sequence of SEQ ID NO:61) using LipofectamineTM2000 Transfection Reagent (Invitrogen, Cat. No. 11668019). A stable single cell clone highly expressing human PD-L1 was selected after screening with puromycin (Gibco, Cat. No. A1113802) to obtain CHO-K1-PDL1 cells.

[0245] Luciferase expression directly correlates with 4-1BB activity.

[0246] HEK293-NFκB-Luc-human 4-1BB cells and A375-PD-L1 cells were harvested and diluted to the appropriate cell density in assay buffer (DMEM + 1% FBS starting at 10 nM, 4 dilutions), respectively, and the two cells were mixed such that the density of HEK293-4-1BB / NFκB cells was 40,000 cells / 60 μL and the density of A375-PD-L1 cells was 20,000 cells / 60 μL. 60 μL / well of the mixture was added to a 96-well plate (Corning, Cat. No. 3917). In a parallel assay testing target cell-independent stimulation, only HEK293-NFκB-Luc-human 4-1BB cells were added to another 96-well plate (Corning, Cat. No. 3917).

[0247] The resulting plate was then added with 60μL / well of serially diluted test samples P4B-3 and INBRX-105-1 (PD-L1x4-1BB bispecific antibody, SEQ ID NO:84, US20170198050A1) (starting at 200nM, 5-fold dilution series, 9 concentrations). After incubation in a 37℃ incubator for 6 hours, 60μL / well of One-Glo (Promega, Cat. No. E6130) reagent was added to the plate. Relative luminescence units were measured with a luminometer (Tecan, Cat. No. F200).

[0248] Figures 9A-B show the ability of PD-L1 x 4-1BB bispecific antibodies (P4B-3 and INBRX-105-1) to induce 4-1BB stimulation without PD-L1. P4B-3 was unable to stimulate the 4-1BB pathway without PD-L1, whereas the INBRX-105-1 control was able to stimulate the pathway at high assay concentrations (Figure 9B).

[0249] HEK293-NFκB-Luc-human 4-1BB cells and CHO-K1-PD-L1 cells were harvested and diluted to the appropriate cell density with assay buffer (DMEM+1%FBS), respectively, and these two cells were mixed. In this case, the density of HEK293-NFκB-Luc-human 4-1BB cells was 40000 cells / 60μL, and the density of CHO-K1-PD-L1 cells was 20000 cells / 60μL. 60μL / well of the mixture was added to a 96-well plate (Corning, Cat. No. 3917).

[0250] Then, 60μL / well of serially diluted test samples P4B-3 and NM21-PRO1186 (PD-L1x4-1BB bispecific antibody, WO2019072868A1) (starting at 200nM, 5-fold dilution series, 9 concentrations) were added to the resulting plate. After 6 hours of incubation in a 37℃ incubator, 60μL / well of One-Glo (Promega, Cat. No. E6130) reagent was added. Relative luminescence units were measured with a luminometer (Tecan, Cat. No. F200).

[0251] Figure 8 shows the ability of PD-L1 x 4-1BB bispecific antibodies (P4B-3 and NM21-PRO1186) to induce PD-L1-binding-dependent 4-1BB stimulation. The results showed that both P4B-3 and NM21-PRO1186 could stimulate the 4-1BB signal pathway in the presence of PD-L1, i.e., dependent on binding to PD-L1. Their agonistic activities are comparable. These results showed that the activation ability of P4B-3 in HEK293-NFκB-Luc-human 4-1BB reporter gene is strictly dependent on binding to PD-L1. Example 9: Activation of human PBMC cells in vitro

[0252] Human peripheral blood mononuclear cells were collected from healthy donors. Mononuclear cells were separated in SepMate 50 tubes (StemCell Technologies) with Lymphoprep density gradient reagent (StemCell Technologies). Both types of cells were frozen in liquid nitrogen for later use.

[0253] 96-well plates (Corning, Cat. No. 3799) were coated overnight with 0.2 μg / mL functional grade anti-CD3 (eBioscience, Cat. No. 16-0037-85) at 4° C. The next day, the coated plates were washed twice with DPBS buffer (Hyclone, SH30256.01).

[0254] PBMC cells (Donor 712023) were rapidly thawed in a 37 °C water bath and the cell suspension was transferred to a tube containing warm complete medium (90% RPMI 1640 (Gibco, Cat. No. 22400) + 10% FBS (Gibco, Cat. No. 10099-141)). The cells were then centrifuged at 300 g for 5 min. The supernatant was discarded and the PBMC cells were resuspended in complete medium (90% RPMI 1640 + 10% FBS) and diluted to 1 × 10 6 Adjusted to cells / mL.

[0255] The target cells Raji / PD-L1 were prepared as follows: Raji parental cell line (Cell Bank, Type Culture Collection, Chinese Academy of Sciences, Catalog No. TCHu44) were infected with human PD-L1 gene (SEQ ID NO:62; encoding the amino acid sequence of SEQ ID NO:61) packaged in a lentivirus system (GeneCopoeiaTM, Catalog No. LT001). To obtain Raji / PD-L1 cells, a stable single cell clone highly expressing human PD-L was selected after screening with puromycin (Gibco, Catalog No. A1113802).

[0256] After incubation, target cells Raji / PD-L1 were harvested by centrifugation at 300g for 5 min. The supernatant was discarded and target cells were diluted to 1 × 10 in complete medium (90% RPMI 1640 + 10% FBS). 6 The antibodies P4B-3, anti-PD-L1 (HUL02 from CN109021107A), 41BB-2-IgG1, anti-PD-L1+41BB-2 (ratio 1:1), and human IgG negative control (heavy chain SEQ ID NO:82, light chain SEQ ID NO:83) prepared in Example 1 were serially diluted to prepare test sample solutions (final concentrations of 0.05 nM, 0.5 nM, 5 nM, and 50 nM).

[0257] Then, 50,000 cells / well of Raji / PD-L1 were added to the coated 96-well assay plate, 50 μL / well of each test sample solution was added, followed by 100,000 cells / well of PBMCs in complete medium. After 72 h of incubation at 37°C and 5% CO2, the cell supernatants were harvested and human interleukin-2 (IL-2) levels in the culture supernatants were quantified using an ELISA kit (R&D, Cat. No. DY202) according to the manufacturer's instructions. The results are shown in Figure 10.

[0258] The results showed that P4B-3 stimulated PBMC cells in a dose-dependent manner to release higher levels of IL-2 than anti-PD-L1 alone, anti-4-1BB (41BB-2-IgG1mut) alone, or the combination of anti-PD-L1 and anti-4-1BB. Example 10: Antitumor effect of P4B-3 in huPD-L1 / hu4-1BB KI mouse model with MC38-hPD-L1

[0259] 5 × 10 6- to 7-week-old BALB / c-huPD-L1 / hu4-1BB double knock-in mice (Biocytogen) 5 MC38-huPD-L1 cells (Biocytogen) were subcutaneously implanted, and the average tumor volume was approximately 87 mm on day 0. 3 (Length x Width 2 When the mean age of patients reached 18.5 years / 2, patients were randomized into four groups.

[0260] Mice were intraperitoneally administered P4B-3, anti-PD-L1 antibody (HUL02 in CN109021107A), 41BB-2-IgG1, and vehicle (PBS) on days 0, 3, 6, 9, 12, and 15. Tumor volumes were monitored by caliper measurements twice weekly throughout the study period.

[0261] Compared with the vehicle group, P4B-3 showed a highly significant tumor-inhibitory effect (P<0.001), with a TGI (tumor growth inhibition) of tumor volume=81.8%, higher than that of the anti-PD-L1 group (TGItv=70.5%) and the anti-4-1BB group (TGItv=46.1%). The mean tumor weight of P4B-3 was also significantly lower than that of the vehicle group (P<0.05).

[0262] Finally, TILs (tumor infiltrating lymphocytes) were isolated from the tumor and immunolabeled with various cell marker antibodies (Brilliant Violet 510 TM Anti-mouse CD45 (Biolegend, Catalog No. 103138), PerCP / Cy5.5 Anti-mouse TCR β chain (Biolegend, Catalog No. 109228), Brilliant Violet 421 TM Anti-mouse CD4 (Biolegend, Cat. No. 100438), Brilliant Violet 711TM Staining was performed with anti-mouse CD8a (Biolegend, Cat. No. 100748), PE anti-mouse / rat Foxp3, eBioscience (Thermo, Cat. No. 12-5773-82)) and then detected by FACS.

[0263] Compared with the vehicle group and anti-PD-L1 group, the proportion of CD8+ T cells in CD3+ T cells in the P4B-3 group was significantly higher (P<0.05), but the proportion of Treg cells in CD3+ cells was significantly lower (P<0.05).

[0264] The results are shown in Figure 11, Figure 12 and Figure 13. These results showed that P4B-3 exhibited strong antitumor effects in the BALB / c-huPD-L1 / hu4-1BB KI mouse model with MC38-huPD-L1 cells. In addition, P4B-3 could also increase the CD8+ ratio and decrease the Treg ratio in the tumor microenvironment. [Table 6] Example 11: Pharmacokinetic study of P4B-3 in rats

[0265] The pharmacokinetic profile of P4B-3 in rats was evaluated. The procedures, including the care and use of animals in the study, were reviewed and approved by PharmaLagancy. Three male SD rats (Shanghai Sippr-BK laboratory animal Co. Ltd.) were used.

[0266] In this study, P4B-3 was intravenously injected into rats at a single dose of 10 mg / kg. Blood samples were collected at various time points from 0 to 336 h (days 0 to 14), specifically at 0 min, 10 min, 30 min, 1 h, 4 h, 8 h on day 0, and on days 1, 2, 4, 7, 10, and 14. All samples were processed to obtain plasma, and the plasma was frozen and stored at -70 to -86 °C for analysis. The concentrations of P4B-3 present in plasma were determined by PD-L1 and 4-1BB antigen capture assays. Pharmacokinetic parameters were shown in Table 7.

[0267] 4-1BB antigen capture assay: Human 4-1BB, human Fc tagged protein (Hu4-1BB-huFc, SEQ ID NO:81) was immobilized on a 96-well plate (Costar, Cat. No. 42592) at 0.5 μg / mL by incubation in carbonate buffer (pH 9.6) overnight at 4°C. The plate was then blocked by incubation with 1% BSA (Sangon Biotech, Cat. No. A500023-0025) in PBS (Hyclone, Cat. No. SH30256.01) for 1 hour at 37°C. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). P4B-3 was diluted at 0.1 μg / mL in serum dilution buffer (PBS containing 0.05% Tween 20 and 0.5% BSA with 2% v / v rat serum) to provide a standard curve with six 3-fold serial dilutions, totaling seven antibody concentrations. At the same time, 80ng / mL, 8ng / mL and 0.8ng / mL of P4B3 were diluted in serum dilution buffer as high, medium and low quality controls, respectively. All rat serum samples were diluted with pre-dosed mixed rat serum and dilution buffer (PBS containing 0.05% Tween20 and 0.5% BSA) to keep the final concentration in the range of 80-0.8ng / mL, and 2% v / v of rat serum was included in the sample dilution. Standard curves, quality controls and samples were added to the plate and incubated at 37°C for 1 h. The plate was then washed three times with PBST and incubated at 37°C for 1 h with peroxidase affinipure goat anti-human IgG, F(ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-035-097) specifically diluted 1 / 10000 in dilution buffer, then washed again with PBST. 50 μL / well of TMB (Thermo, Cat. No. 34028) was added to the plate, and after 15 minutes, the reaction was stopped with 1M H2SO4. The absorbance was measured at 450 nm to 620 nm.

[0268] PD-L1 antigen capture assay: Human PD-L1, His tagged protein (huPD-L1-his, ACRO, Cat. No. 10084-H08H) was immobilized on a 96-well plate (Costar, Cat. No. 42592) at 0.5μg / mL by incubation in carbonate buffer (pH 9.6) overnight at 4℃. The plate was then blocked by incubating with 1% BSA in PBS at 37℃ for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween20). P4B-3 was diluted to 0.1μg / mL in serum dilution buffer (PBS containing 0.05% Tween20 and 0.5% BSA with 2% v / v rat serum), and six 3-fold serial dilutions, totaling seven concentrations of antibody solution, were used as the standard curve. At the same time, 80ng / mL, 8ng / mL and 0.8ng / mL of P4B3 were diluted in serum dilution buffer as high, medium and low quality controls, respectively. All rat serum samples were diluted with pre-dosed mixed rat serum and dilution buffer (PBS containing 0.05% Tween20 and 0.5% BSA) to keep the final concentration in the range of 80-0.8ng / mL, and 2% v / v of rat serum was included in the sample dilution. Standard curves, quality controls and samples were added to the plate and incubated for 1 h at 37°C. The plate was then washed three times with PBST and incubated for 1 h at 37°C with peroxidase affinipure goat anti-human IgG, Fc fragment (Jackson ImmunoResearch, Cat. No. 109-035-098) specifically diluted 1 / 20000 in dilution buffer, then washed again with PBST. 50 μL / well of TMB (Thermo, Cat. No. 34028) was added to the plate, and after 15 minutes, the reaction was stopped with 1M H2SO4. The absorbance was measured at 450 nm to 620 nm. [Table 7]

[0269] The pharmacokinetics of P4B-3 was favorable in rats, indicating that it could be used in pharmaceutical fields.

[0270] While the present disclosure has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents falling within the spirit and scope of the appended claims. All references cited herein are further incorporated by reference in their entirety. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

Claims

Claim 1: A bispecific antibody comprising a first antigen-binding region and a second binding region, the first binding domain binds to 4-1BB; The first binding region comprises a heavy chain variable region CDR1 region, a CDR2 region, and a CDR3 region, and a light chain variable region CDR1 region, a CDR2 region, and a CDR3 region. Including, the heavy chain variable region CDR1 region, CDR2 region, and CDR3 region comprise the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region CDR1 region, CDR2 region, and CDR3 region comprise the sequences shown in SEQ ID NOs: 4, 5, and 6, respectively; Bispecific antibodies.

2. A bispecific antibody comprising a heavy chain variable region, 2. The bispecific antibody of claim 1, wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25 or 77.

3. A bispecific antibody comprising a light chain variable region, 3. The bispecific antibody of claim 1 or 2, wherein the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26 (X1 = S or G) or 78.

4. (1) a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 26; or (2) A heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 77 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

78.

2. The bispecific antibody of claim 1 , comprising:

5. A bispecific antibody according to any one of claims 1 to 4, wherein the first antigen-binding region and / or the second binding region is a mouse, human, chimeric or humanized antibody.

6. The bispecific antibody of any one of claims 1 to 5, wherein the first antigen-binding region and / or the second binding region is selected from (i) a Fab fragment, (ii) an F(ab')2 fragment, (iii) an Fd fragment, (iv) an Fv fragment, (v) a dAb fragment, (vi) a nanobody, or (vii) a single-chain Fv (scFv).

7. The bispecific antibody of claim 1, wherein the second antigen is selected from PD-L1, PD1, or CTLA-4.

8. The method of claim 7, wherein the second antigen is PD-L1, and the antibody against PD-L1 comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three heavy chain variable regions CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 consist of the sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively; The light chain variable region comprises three light chain variable regions CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 consist of the sequences set forth in SEQ ID NOs: 43, 44 and 45, respectively; The bispecific antibody of claim 7.

9. The first antigen-binding region is an scFv, and the scFv of the anti-4-1BB antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three heavy chain variable regions CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise the sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively; The light chain variable region comprises three light chain variable regions CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise the sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively; The bispecific antibody according to any one of claims 1 to 8.

10. Chain 1 of the bispecific antibody comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 48 or 50; and Chain 2 of the bispecific antibody comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:

49. The bispecific antibody according to any one of claims 1 to 9.

11. A polynucleotide encoding a bispecific antibody or a chain thereof according to any one of claims 1 to 10.

12. A vector comprising the polynucleotide described in claim 11.

13. A host cell having a genome integrated with the polynucleotide described in claim 11, or containing the vector described in claim 12, or containing the polynucleotide described in claim 11.

14. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

15. A combination product comprising the bispecific antibody of any one of claims 1 to 10 and one or more additional therapeutic agents.

16. The combination product of claim 15, wherein the additional therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a vaccine, another antibody, an anti-infective agent, a small molecule drug, or an immunomodulatory agent.

17. The combination product described in claim 16, wherein the other antibody is selected from antibodies against immune checkpoint molecules.

18. The combination product described in claim 17, wherein the immune checkpoint molecule is PD-L1 or PD-1.

19. A pharmaceutical composition according to claim 14, or a combination product according to any one of claims 15 to 18, for treating cancer in a subject, or for treating an infectious disease in a subject, or for treating an autoimmune disease in a subject.

20. The pharmaceutical composition or combination product of claim 19, wherein the cancer is tumor immune evasion.

21. The pharmaceutical composition or combination product of claim 19, wherein the cancer is selected from a solid cancer, a non-solid cancer, and a metastatic lesion, and the cancer is a malignant tumor.

22. The pharmaceutical composition or combination of claim 19, wherein the cancer is a cancer having an increased level of PD-L1 protein expression or an increased level of nucleic acid encoding PD-L1, e.g., compared to levels in normal subjects or normal cells.

23. The pharmaceutical composition or combination of claim 19, wherein the cancer is cancer in the digestive tract (gastrointestinal tract), colorectal cancer (large intestine cancer), rectal cancer, or colon cancer.

24. The pharmaceutical composition or combination of any one of claims 19 to 23, wherein the subject can be treated with a therapeutic modality and / or other therapeutic agent, the therapeutic modality comprising surgery and / or radiation therapy, and / or the other therapeutic agent is selected from the group consisting of a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulatory agent.

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