CD137 agonist antibodies and uses thereof

Abstract

The present disclosure provides isolated binding molecules that bind to and activate CD137, vectors comprising nucleic acid molecules encoding the amino acid sequence of the binding molecules, host cells containing the vectors, methods of making the binding molecules, pharmaceutical compositions containing the binding molecules, and methods of using such antibodies, antibody fragments and derivatives, and polypeptides, methods of treating diseases requiring stimulation of an immune response, including cancer, are included.

Description

[0001] This application is a divisional application of a patent application with an application date of January 2, 2020, application number 202080012041.6, and name “CD137 agonist antibodies and their uses”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 787,509, filed on January 2, 2019, the entire disclosure of which is incorporated herein by reference.

[0004] Incorporation of a Sequence Listing This application includes a sequence listing named "QLSF001PCT_ST25.txt" with a size of 73.8 KB, created on January 2, 2020 and submitted in ASCII format via EFS-Web. The entire contents of the sequence listing are incorporated herein by reference. Background of the Invention

[0006] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its aliases are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, and receptor for lymphocyte activation induction (ILA). CD137 is expressed by activated T cells, but the expression level on CD8 T cells is higher than that on CD4 T cells. In addition, CD137 is expressed on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. A typical activity of CD137 is its co-stimulatory activity on activated T cells. Cross-linking of CD137 can enhance T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In addition, it can enhance immune activity to eliminate tumors in mice.

[0007] CD137 can be induced as a T cell co-stimulatory receptor upon TCR activation (Nam et al., Current Cancer Drug Targets, 5: 357-363 (2005); Watts et al., Annual Review of Immunology, 23: 23-68 (2005)). In addition to being expressed on activated CD4+T cells and CD8+T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells. Its natural ligand CD137L is expressed on antigen presenting cells, including B cells, monocytes / macrophages and dendritic cells (Watts et al., Annu. Rev. Immunol., 23: 23-68 (2005)).

[0008] CD137L or CD137 agonistic monoclonal antibodies (mAbs) enhance TCR-induced T cell proliferation, cytokine production, and functional maturation and prolong CD8+ T cell survival through CD137 signaling. These effects result from: (1) activation of NF-KB, c-JunNH2-terminal kinase / stress-activated protein kinase (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and (2) regulation of anti-apoptotic and cell cycle-related gene expression. Experiments in mice lacking CD137 and CD137L also demonstrate the importance of CD137 co-stimulation in generating fully competent T cell responses. IL-2 and IL-15-activated NK cells express CD137, and agonistic mAb ligation of CD137 stimulates NK cell proliferation and IFN-γ secretion, but not cytolytic activity. In addition, CD137-stimulated NK cells promote the expansion of activated T cells in vitro. Based on its co-stimulatory function, agonistic mAbs against CD137 have been shown to promote rejection of cardiac allografts and skin allografts, eradicate tumors, expand primary antiviral CD8+ T cell responses, and enhance the cytolytic potential of T cells. These studies support the concept that CD137 signaling promotes T cell function, which can enhance immunity to tumors and infections.

[0009] Anti-CD137 antibodies have been disclosed in U.S. 2005 / 0095244, U.S. Pat. No. 7,288,638 (e.g., 20H4.9-IgG4 [10C7 or BMS-663513] or 20H4.9-IgG1 [BMS-663031]); U.S. Pat. No. 6,887,673 [cxE9 or BMS-554271]; U.S. Pat. No. 7,214,493; U.S. Pat. No. 6,303,121; U.S. Pat. No. No. 6,569,997; U.S. Patent No. 6,905,685; U.S. Patent No. 6,355,476; U.S. Patent No. 6,362,325 [IDS or BMS-469492; 3H3 or BMS-469497; or 3E1]; U.S. Patent No. 6,974,863 (e.g., 53A2); or U.S. Patent No. 6,210,669 (e.g., IDS, 3B8 or 3E1), or U.S. Patent No. 8,337,850. Additional CD137 agonistic antibodies are described in U.S. 2016 / 0244528, U.S. Patent No. 5,928,893; U.S. Patent No. 6,303,121; U.S. Patent No. 6,569,997 and U.S. Patent No. 8,137,667; SUMMARY OF THE INVENTION

[0011] The present disclosure provides isolated anti-CD137 monoclonal agonist antibodies and antigen-binding portions thereof that specifically bind to human CD137.

[0012] In one aspect of the invention, an isolated anti-CD137 monoclonal agonist antibody or antigen binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 8. In some embodiments, the anti-CD137 monoclonal agonist antibody or antigen binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 6, and a heavy chain variable region CDR2 comprising SEQ ID NO: 7. In a preferred embodiment, the anti-CD137 monoclonal agonist antibody or antigen binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 3; (b) a light chain variable region CDR2 comprising SEQ ID NO: 4; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 5. In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:1 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:2. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO:2.

[0013] In one aspect of the invention, an isolated anti-CD137 monoclonal agonist antibody or antigen binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 31. In some embodiments, the anti-CD137 monoclonal agonist antibody or antigen binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 29, and a heavy chain variable region CDR2 comprising SEQ ID NO: 30. In a preferred embodiment, the anti-CD137 monoclonal agonist antibody or antigen binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 26; (b) a light chain variable region CDR2 comprising SEQ ID NO: 27; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 28. In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:35 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:36. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO:36.

[0014] In another aspect of the invention, an isolated anti-CD137 monoclonal agonist antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22 to SEQ ID NO:25; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:18 to SEQ ID NO:21.

[0015] In another aspect of the invention, an isolated anti-CD137 monoclonal agonist antibody, or an antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 12.

[0016] The antibodies of the disclosed invention can be further engineered into a form suitable for human treatment by modifications that minimize immunogenicity. Suitable antibodies include, but are not limited to, chimeric antibodies and humanized antibodies. The affinity, stability and specificity of the disclosed antibodies can also be further optimized by techniques known to those skilled in the art. Other forms may involve oligomerization, drug conjugation and fusion of the disclosed antibodies with other functional proteins.

[0017] The antibodies of the disclosed invention may be, for example, full-length antibodies, such as IgG1, IgG2, IgG3 or IgG4 isotypes. Alternatively, the disclosed antibodies may be antibody fragments, such as Fab fragments, Fab' fragments and F(ab') fragments. 2 Fragments, diabodies, trimers, tetrabodies, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv) and half antibodies. Alternatively, the disclosed antibodies may be bispecific antibodies.

[0018] In another aspect of the invention, an isolated monoclonal antibody, or an antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:37 to SEQ ID NO:39; and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:40 to SEQ ID NO:42.

[0019] In another aspect of the invention, an isolated monoclonal antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:46; and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:47 to SEQ ID NO:50.

[0020] In some embodiments, the anti-CD137 agonist antibody or its antigen binding portion binds to human CD137 and activates it. Therefore, the antibody or antigen binding portion can stimulate an anti-tumor immune response. In some embodiments, the anti-CD137 agonist antibody or its antigen binding portion binds to non-human primate CD137 and activates it. In some embodiments, the anti-CD137 agonist antibody or its antigen binding portion binds to mammalian CD137 and activates it.

[0021] In another aspect of the present invention, a composition comprising the isolated anti-CD137 monoclonal agonist antibody or an antigen-binding portion thereof is also provided.

[0022] In another aspect of the present invention, a pharmaceutical composition comprising the isolated anti-CD137 monoclonal agonist antibody or its antigen binding portion and a pharmaceutically acceptable transporter is also provided. A composition comprising the immunoconjugate of the present invention and a pharmaceutically acceptable transporter is also provided.

[0023] In another aspect of the present invention, a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof, and a host cell comprising an expression vector comprising the nucleic acid molecule are also provided.

[0024] The present invention further provides a method of stimulating an immune response using the anti-CD137 agonist antibodies of the present disclosure. For example, in one embodiment, the present disclosure provides a method of treating a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding portion of the present disclosure.

[0025] In another aspect, the present disclosure provides a method of treating cancer in a human, comprising the step of administering to the human an amount of an anti-CD137 agonist antibody or antigen-binding portion of the present disclosure effective to treat the cancer.

[0026] In another aspect, the present disclosure provides a method of treating an infectious disease in a human, comprising the step of administering to the human an amount of an anti-CD137 agonist antibody or antigen binding portion of the present disclosure effective to treat the infectious disease.

[0027] Other features and advantages of the present disclosure will be apparent from the following description and examples, which should not be construed as limiting.The contents of all references, GenBank entries, patents, and published patent applications cited in this application are expressly incorporated herein by reference.

[0028] The present application also includes the following implementation modes.

[0029] 1. An isolated anti-CD137 agonist antibody or an antigen-binding portion thereof, comprising a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises SEQ ID NO: 8.

[0030] 2. An isolated anti-CD137 agonist antibody or an antigen-binding portion thereof, comprising a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises SEQ ID NO:31.

[0031] 3. The isolated antibody or antigen-binding portion thereof according to embodiment 1, further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 6, and a heavy chain variable region CDR2 comprising SEQ ID NO: 7, respectively.

[0032] 4. The isolated antibody or antigen-binding portion thereof according to embodiment 2 further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 29, and a heavy chain variable region CDR2 comprising SEQ ID NO: 30, respectively.

[0033] 5. The isolated antibody or antigen-binding portion thereof according to embodiment 3, further comprising

[0034] (a) a light chain variable region CDR1 comprising SEQ ID NO: 3;

[0035] (b) a light chain variable region CDR2 comprising SEQ ID NO: 4; and

[0036] (c) a light chain variable region CDR3 comprising SEQ ID NO: 5;

[0037] Wherein, the antibody or portion specifically binds to human CD137.

[0038] 6. The isolated antibody or antigen-binding portion thereof according to embodiment 4, further comprising

[0039] (a) a light chain variable region CDR1 comprising SEQ ID NO: 26;

[0040] (b) a light chain variable region CDR2 comprising SEQ ID NO: 27; and

[0041] (c) a light chain variable region CDR3 comprising SEQ ID NO: 28;

[0042] Wherein, the antibody or portion specifically binds to human CD137.

[0043] 7. An isolated antibody or antigen-binding portion thereof according to embodiment 1, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:1 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:2.

[0044] 8. According to the isolated antibody or its antigen-binding portion described in embodiment 1, it comprises a heavy chain variable region, which comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO:2.

[0045] 9. An isolated antibody or antigen-binding portion thereof according to embodiment 2, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:35 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:36.

[0046] 10. According to the isolated antibody or its antigen-binding portion described in embodiment 2, it comprises a heavy chain variable region, which comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO:36.

[0047] 11. The isolated antibody or antigen-binding portion thereof according to any one of embodiments 1-10, which is a Fab fragment, F(ab') 2 fragment, Fv fragment, single chain antibody or bispecific antibody.

[0048] 12. According to any one of embodiments 1-10, the isolated antibody is a chimeric antibody or a humanized antibody.

[0049] 13. An isolated antibody according to any one of embodiments 1-10, wherein the isolated antibody is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule.

[0050] 14. According to the isolated antibody described in embodiment 13, the isolated antibody is IgG1, IgG2, IgG3 or IgG4.

[0051] 15. According to embodiment 1, the isolated antibody or its antigen-binding portion comprises: a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 13 to SEQ ID NO: 17, and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 9 to SEQ ID NO: 12.

[0052] 16. The isolated antibody or antigen-binding portion thereof according to embodiment 2 comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:22 to SEQ ID NO:25; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:18 to SEQ ID NO:21.

[0053] 17. According to any one of embodiments 15 and 16, the isolated antibody is a chimeric antibody or a humanized antibody.

[0054] 18. An isolated antibody according to any one of embodiments 15 and 16, wherein the isolated antibody is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule.

[0055] 19. According to the isolated antibody described in embodiment 18, the isolated antibody is IgG1, IgG2, IgG3 or IgG4.

[0056] 20. An isolated antibody according to any one of embodiments 1-19, wherein the antibody is a monoclonal antibody.

[0057] 21. An isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:37 to SEQ ID NO:39; and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:40 to SEQ ID NO:42.

[0058] 22. An isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:46; and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:47 to SEQ ID NO:50.

[0059] 23. An immunoconjugate comprising the antibody or antigen-binding portion thereof according to any one of embodiments 1-10, 15, 16, 21 and 22 linked to a therapeutic agent.

[0060] 24. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of embodiments 1-10, 15, 16, 21 and 22 and a pharmaceutically acceptable transporter.

[0061] 25. A method for stimulating an immune response in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to embodiment 24 to stimulate the immune response in the subject.

[0062] 26. A method for treating an infectious disease in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to embodiment 24 to treat the infectious disease.

[0063] 27. A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to embodiment 24 to treat the cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Exemplary embodiments are shown in the referenced drawings.The embodiments and drawings disclosed herein are to be considered for illustrative rather than restrictive purposes.

[0065] Figure 1A and Figure 1B It is shown that the anti-4-1BB (anti-CD137) agonist antibodies of the present disclosure induce NF-KB reporter gene activation. Figure 2A and Figure 2B It is shown that the anti-4-1BB (anti-CD137) agonist antibodies of the present disclosure stimulate proliferation of primary CD3+CD8+ T cells. Figure 2C and Figure 2D Anti-4-1BB (anti-CD137) agonist antibodies that stimulate proliferation of primary CD3+CD8- T cells are shown.

[0066] Figure 3A and Figure 3B The anti-4-1BB (anti-CD137) agonist antibodies of the present disclosure are shown to stimulate interferon-γ secretion.

[0067] Figure 4A and Figure 4B The anti-4-1BB (anti-CD137) agonist antibodies of the present disclosure were shown to inhibit cancer in hu4-1BB knock-in mice. DETAILED DESCRIPTION OF THE INVENTION

[0069] The embodiments and aspects thereof are described and illustrated below in conjunction with systems, compositions, and methods which are for exemplary and illustrative purposes only and not limiting in scope.

[0070] definition

[0071] As used herein, the terms "include" or "comprising" are used to indicate compositions, methods and their respective components that are useful for the embodiments but may include unspecified elements (whether useful or not). Those skilled in the art will understand that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "include" should be interpreted as "including but not limited to", the term "have" should be interpreted as "have at least", the term "include" should be interpreted as "include but not limited to", etc.).

[0072] Unless otherwise stated, the terms "a", "an" and "said" and similar quotations used in the context of describing a specific embodiment of the present application (especially in the context of the claims) may be interpreted as covering the singular and plural. References to numerical ranges herein are intended only to be used as a shorthand method of referring to each individual value falling within the range individually. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually quoted herein. Unless otherwise stated herein or clearly contradictory to the context, all methods described herein may be performed in any suitable order. The use of all examples or exemplary language (e.g., "such as") associated with certain embodiments herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application to which protection is otherwise claimed. The abbreviation "eg" is derived from Latin exempli gratia and is used herein to indicate non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the word "for example". Any language in this specification should not be interpreted as representing any unclaimed element that is essential to the practice of the present application.

[0073] As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and encompasses variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified value.

[0074] As used herein, the term "epitope" may include any protein determinant that can specifically bind to an immunoglobulin or T cell receptor. Epitope determinants are usually composed of chemically active molecular surface groups, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. When the equilibrium dissociation constant is ≤1 μM (preferably ≤100nM, most preferably ≤10nM), the antibody is said to specifically bind to the antigen.

[0075] The term "K D ” may refer to the equilibrium dissociation constant for a specific antibody-antigen interaction.

[0076] As used herein, the term "immune response" may refer to, for example, the actions of lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which can cause selective damage to the organism, destroy or eliminate invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal body cells or tissues (in the case of autoimmunity or pathological inflammation) in the organism.

[0077] As used herein, "antigen-specific T cell response" may refer to a T cell response caused by stimulating T cells with a T cell-specific antigen. Non-limiting examples of T cell responses to antigen-specific stimulation include proliferation and production of cytokines (eg, production of IL-2).

[0078] As used herein, the term "antibody" refers to an intact immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having an Fc (fragment crystallizable) region or an FcRn binding fragment of an Fc region, referred to herein as an "Fc fragment" or "Fc region". Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, inter alia, Fab, Fab', F(ab') 2 , Fv, dAb and complementary determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigens bound to the polypeptide. The Fc region includes portions of the two heavy chains that constitute two or three classes of antibodies. The Fc region can be produced by recombinant DNA technology or by enzymatic (e.g., papain cleavage) or by chemical cleavage of intact antibodies.

[0079] As used herein, the term "antibody fragment" refers to a protein fragment that contains only a portion of an intact antibody, generally including the antigen binding site of the intact antibody and thus retaining the ability to bind to an antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having a VL region, a CL region, a VH region, and a CH1 region; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 region; (iii) a Fd fragment having a VH region and a CH1 region; (iv) a Fd' fragment having a VH region and a CH1 region and one or more cysteine ​​residues at the C-terminus of the CH1 region; (v) a Fv fragment having a single-arm VL region and a VH region of an antibody; (vi) a dAb fragment consisting of a VH region (Ward et al., Nature 341, 544-546 (1989)); (vii) an isolated CDR region; (viii) a F(ab') 2(ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen-binding sites, comprising a heavy chain variable region (VH) connected to a light chain variable region (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Natl. Acad. Sci. 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0080] As used herein, "single chain variable fragment", "single chain antibody variable fragment" or "scFv" antibody refers to an antibody format containing only the variable regions of the heavy chain (VH) and the light chain (VL) connected by a linker peptide. scFv can be expressed as a single chain polypeptide. scFv retains the specificity of the intact antibody from which it is derived. The light chain and heavy chain can be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is maintained.

[0081] As used herein, "isolated antibodies" may refer to antibodies that are substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to a CD137 protein may be substantially free of antibodies that specifically bind to antigens other than the CD137 protein). However, an isolated antibody that specifically binds to a human CD137 protein may have cross-reactivity with other antigens (such as CD137 proteins from other species). In addition, the isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0082] Anti-CD137 agonist antibody producing cells, such as hybridomas, can be selected, cloned and further screened to obtain desired characteristics, including robust growth, high antibody production and desired antibody characteristics. Hybridomas can be amplified in isogenic animals, animals lacking an immune system (e.g., nude mice) or in vitro cell cultures. Methods for selecting, cloning and amplifying hybridomas are well known to those of ordinary skill in the art.

[0083] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein may refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0084] As used herein, the term "recombinant human antibody" may refer to all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (as described below), (b) antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfectomas (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, may not exist in the human antibody germline library in vivo under natural conditions.

[0085] The term "isotype" may refer to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The antibody may be an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule, or derived from them.

[0086] Herein, the phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably with the term "an antibody that specifically binds to an antigen."

[0087] As used herein, an antibody that "specifically binds to human CD137" may refer to an antibody that binds to human CD137 protein (and CD137 protein that may be from one or more non-human species) but does not substantially bind to non-CD137 proteins. Preferably, the antibody binds to human CD137 protein with "high affinity", i.e., with a affinity of 1x10 -7 M or less, more preferably 5x10 -8 M or less, more preferably 3x10 -8 M or less, more preferably 1x10 -8 M or less, more preferably 5x10 -9 M or less or even more preferably 1x10 -9 M or less K D .

[0088] As used herein, the term "does not substantially bind" a protein or cell may mean that it is unable to bind or does not bind with high affinity to a protein or cell, i.e., with an affinity of 2×10 -6 M or more, more preferably 1x10 -5 M or more, more preferably 1x10 -4 M or more, more preferably 1x10 -3 M or more, even more preferably 1x10 -2 M or above K D Bind to proteins or cells.

[0089] The term "high affinity" for IgG antibodies may refer to antibodies with a 1x10 -6 M or less, preferably 1x10 -7 M or less, more preferably 1x10 -8 M or less, even more preferably 1x10 -9 M or less, even more preferably 1x10 -10 M or less K D However, "high affinity" binding can vary for other antibody isotypes.

[0090] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredients contained therein to be effective and which contains no additional ingredients which would be unacceptably toxic to a subject to which the preparation would be administered.

[0091] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical preparation or cell) refers to an effective amount that achieves a desired therapeutic outcome, such as a pharmacokinetic or pharmacodynamic effect for treating a disease, condition, or disorder and / or a therapeutic effect, within the necessary dosage and time period. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population being administered. In some embodiments, the methods provided herein involve administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0092] As used herein, "agonist antibodies" are antibodies that induce or increase the biological activity of an antigen (e.g., CD137) to which the antibody binds. For example, due to the binding of the receptor to the ligand, the agonist can promote the phosphorylation of the receptor, or can activate or grow cells activated by the receptor. In one embodiment, the antibody of the invention is an anti-CD137 agonist antibody.

[0093] A "CDR-grafted antibody" is an antibody that comprises one or more CDRs derived from a particular species or isotype and the framework of another antibody of the same or different species or isotype.

[0094] "Humanized antibodies" have sequences that are different from antibodies derived from non-human species by one or more amino acid substitutions, deletions and / or additions, so that when administered to human subjects, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response than non-human species antibodies. In one embodiment, certain amino acids in the framework and constant region of the heavy chain and / or light chain of non-human species antibodies are mutated to produce humanized antibodies. In another embodiment, the constant region from a human antibody is fused with the variable region of a non-human species. In another embodiment, the humanized antibody is a CDR-grafted antibody that comprises one or more CDRs derived from antibodies of a specific species or isotype and a human antibody framework. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the possible immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly inferior to the binding of the non-human antibody to the antigen. For examples of how to make humanized antibodies, see U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0095] The term "chimeric antibody" refers to an antibody comprising one or more regions from an antibody and one or more regions from one or more other antibodies. In one embodiment, one or more CDRs are derived from human anti-CD137 antibodies. In another embodiment, all CDRs are derived from human anti-CD137 antibodies. In another embodiment, CDRs from more than one human anti-CD137 antibodies are mixed and matched in a chimeric antibody. For example, a chimeric antibody may include CDR1 from a light chain of a first human anti-CD137 antibody, CDR2 and CDR3 from a light chain of a second human anti-CD137 antibody, and CDRs from a heavy chain of a third anti-CD137 antibody. Other combinations are also possible.

[0096] The term "subject" may refer to any human or non-human animal. The subject may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and elderly subjects. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, rabbits, mice, rats, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0097] The binding of the antibodies of the present disclosure to CD137 can be evaluated using one or more mature techniques in the art. For example, in a preferred embodiment, the antibodies can be tested by ELISA assays, for example using recombinant CD137 protein. Other suitable binding assays include, but are not limited to, flow cytometry assays, in which the antibodies react with cell lines expressing human CD137, such as HEK293 cells that have been transfected to express CD137 (e.g., human CD137) on their cell surfaces. In addition or alternatively, the binding of the antibodies can be tested in BIAcore binding assays, OctetRed96 (Pall), etc., including binding kinetics (e.g., K D value).

[0098] Preferably, the antibodies of the present invention bind to 5x10 -8 M or less K D Human CD137 protein, binding with 2x10 -8 M or less K D Human CD137 protein, binding with 2x10 -8 M or less K D Human CD137 protein, binding with 5x10 -9 M or less K D Human CD137 protein, binding with 4x10 -9 M or less K D Human CD137 protein, binding with 3x10 -9 M or less K D Human CD137 protein, binding with 2x10 -9 M or less K D Human CD137 protein, binding with 1x10 -9 M or less K D of human CD137 protein.

[0099] The present disclosure relates to isolated monoclonal antibodies or antigen-binding portions thereof that bind to and activate CD137 and their uses. In certain embodiments, the antibodies of the present disclosure are derived from identified heavy and light chain germline sequences and / or contain identified structural features, such as CDR regions containing identified amino acid sequences. The present disclosure provides isolated antibodies of the present disclosure, methods for preparing such antibodies, and antigen-binding portions thereof. The present disclosure also relates to methods of using antibodies, such as using the anti-CD137 agonist antibodies of the present disclosure alone or in combination with other immunostimulatory antibodies to stimulate immune responses. Therefore, methods of using the anti-CD137 agonist antibodies of the present disclosure are also provided, for example, including but not limited to treating human cancers. Various aspects of the present invention relate to antibodies and antibody fragments, pharmaceutical compositions, nucleic acids, recombinant expression vectors, and host cells for preparing such antibodies and fragments. The present invention also includes methods of detecting human CD137, stimulating CD137 activity in vitro or in vivo, and preventing or treating diseases such as cancer using the antibodies of the present invention.

[0100] Complementarity determining regions (CDRs) are referred to as hypervariable regions in the light chain variable region and the heavy chain variable region. The more highly conserved portions of the variable region are referred to as frameworks (FRs). The complementary determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using the systems described by Kabat et al., Lefranc et al., and / or Honegger and Pluckthun above. Those skilled in the art are also familiar with the numbering system described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). In this regard, Kabat et al. define a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region amino acid sequence without relying on any experimental data other than the sequence itself.

[0101] In certain embodiments, the present invention provides an anti-CD137 agonist antibody or an antigen-binding portion thereof. In one embodiment, the antibody or portion comprises (a) a light chain variable region CDR1 comprising SEQ ID NO: 3; (b) a light chain variable region CDR2 comprising SEQ ID NO: 4; (c) a light chain variable region CDR3 comprising SEQ ID NO: 5; (d) a heavy chain variable region CDR1 comprising SEQ ID NO: 6; (e) a heavy chain variable region CDR2 comprising SEQ ID NO: 7; (f) a heavy chain variable region CDR3 comprising SEQ ID NO: 8. In another embodiment, the antibody or portion comprises (a) a light chain variable region CDR1 comprising SEQ ID NO:26; (b) a light chain variable region CDR2 comprising SEQ ID NO:27; (c) a light chain variable region CDR3 comprising SEQ ID NO:28; (d) a heavy chain variable region CDR1 comprising SEQ ID NO:29; (e) a heavy chain variable region CDR2 comprising SEQ ID NO:30; (f) a heavy chain variable region CDR3 comprising SEQ ID NO:31.

[0102] In one embodiment, the present disclosure provides a monoclonal antibody, or antigen binding portion thereof, that binds to a CD137 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 35 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 36.

[0103] Given that each of these antibody Fabs can bind to human CD137, VH sequences and VL sequences can be "mixed and matched" to produce other anti-CD137 binding molecules of the present invention. Preferably, when VH chains and VL chains are mixed and matched, the VH sequence from a specific VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, preferably, the VL sequence from a specific VH / VL pairing is replaced with a structurally similar VL sequence.

[0104] In some embodiments, the humanized antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 12. Preferred heavy chain and light chain combinations include, but are not limited to:

[0105] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

[0106] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10;

[0107] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11;

[0108] (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12;

[0109] In some embodiments, the humanized antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 25, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 21. Preferred heavy chain and light chain combinations include, but are not limited to:

[0110] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18;

[0111] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19;

[0112] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20;

[0113] (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21;

[0114] In some embodiments, the humanized anti-CD137 agonist antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 37 to SEQ ID NO: 39, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 40 to SEQ ID NO: 42.

[0115] In some embodiments, the humanized anti-CD137 agonist antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43 to SEQ ID NO:46, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:47 to SEQ ID NO:50.

[0116] In some embodiments, the present invention provides an anti-CD137 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in any one of SEQ ID NO: 8 and SEQ ID NO: 31, and comprising a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 13 to SEQ ID NO: 17, SEQ ID NO: 22 to SEQ ID NO: 25, and SEQ ID NO: 36.

[0117] In some embodiments, the present invention provides an anti-CD137 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in any one of SEQ ID NO:5 and SEQ ID NO:28, and having a light chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO:1, SEQ ID NO:9 to SEQ ID NO:12, SEQ ID NO:18 to SEQ ID NO:21 and SEQ ID NO:35.

[0118] Thus, in certain embodiments, the CDR3 region remains constant, while variability can be introduced into the remaining CDR and / or framework regions of the heavy and / or light chain, while the antibody or antigen-binding fragment thereof retains the ability to bind to CD137 and retains the functional properties of the parent, such as binding affinity.

[0119] In some embodiments, substitutions made within heavy or light chains that are at least 95% identical (or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) are conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Where two or more amino acid sequences differ from one another by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, tryptophan; (5) basic side chains: lysine, arginine, histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine.

[0120] The present disclosure also provides isolated polynucleotides (or nucleic acid molecules) encoding various amino acid sequences disclosed herein (e.g., amino acid sequences of antibodies or antigen-binding portions thereof), vectors comprising the polynucleotides, cells (or host cells) comprising the vectors, cells comprising or expressing the various amino acid sequences disclosed herein, methods for preparing antibodies (and / or fragments thereof), pharmaceutical compositions comprising the antibodies disclosed herein, and the like.

[0121] Unless otherwise specified or implied from the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated, or obvious from the context, the following terms and phrases do not exclude the meanings that the term or phrase has acquired in the art to which it belongs. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs.

[0122] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication referenced, whether expressly or implicitly, is prior art.

[0123] Working Example:

[0124] The following examples are not intended to limit the scope of the claims of the present invention, but are intended as examples of certain embodiments. Any variations in the example methods that occur to a skilled person are intended to fall within the scope of the present invention.

[0125] Vector construction:

[0126] The vector pcDNA3.4TOPO (Invitrogen) was connected to a short polylinker comprising EcoRI, XhoI and NotI. The resulting plasmid was digested with EcoRI and NotI restriction enzymes and purified by gel electrophoresis. For heavy chain cloning, the prepared vector was assembled using the Gibson assembly method, and the prepared vector, the gblock encoding the VH region (IDT) and the gblock encoding human IgG2 with an XhoI endonuclease site added at the junction of the J chain and CH1 region were assembled. The plasmid was prepared and digested with EcoRI and XhoI to adapt to all humanized heavy chain variable regions (VH) with IgG2 isotypes. All assemblies were completed by the Gibson method (NEB). The light chain variable region was constructed by a similar method and using gblocks to assemble the Vkappa region together by the gblock fragment encoding constant kappa (Ck).

[0127] Protein expression:

[0128] Plasmids were prepared and transfected into Expi293 or ExpiCHO cells using a transient expression system (ThermoFisher). Briefly, plasmids were transfected into 3e6 cells / mL of cells at 1 μg total plasmid DNA / mL culture. Heavy and light chain plasmids were mixed in a 1:1 ratio. The cultures were incubated with shaking at 37°C. After 16 hours, Transfection Enhancer 1 and Transfection Enhancer 2 were added to the culture and incubated for another 6 days. The supernatant was filtered and the protein titer was determined using OctetRed96 (Pall) using an IgG quantification protocol. (Table 1) IgG was purified by Mab Select Sure Protein-A column purification on the ACTA PURE system and dialyzed overnight in PBS.

[0129] Table 1: Octet data. Monovalent binding kinetics of anti-4-1BB antibodies

[0130]

[0131] Flow cytometry

[0132] HEK293 cells transfected with human / cynomolgus monkey / mouse 4-1BB or PBMC stimulated with PHA-P were incubated with serially diluted anti-4-1BB antibodies in FACS buffer (DPBS + 2% FBS + 0.05% sodium azide) and subsequently labeled with AF647 F(ab') 2 Goat anti-human IgG and 7-AAD. In addition, PBMCs were incubated with FITC mouse anti-human CD3. Samples stained for HEK293 expressing 4-1BB were analyzed using FlowJo by gating on FSC / SSC followed by live / dead cell gating, and human / cynomolgus / mouse 4-1BB+ cells (expressed as MFI Geo average). Samples stained for lymphocytes were analyzed using FlowJo by gating on FSC / SSC followed by live / dead cell gating, and CD3+ cells with 4-1BB positivity. (Tables 2 and 3)

[0133] Table 2: Flow cytometry and NK-kB luciferase assays. Anti-4-1BB mAb binds to cell surface antigens. Anti-4-1BB mAb does not bind to mouse 4-1BB or unstimulated human T cells (data not shown).

[0134] In the absence of Fc cross-linking, no NF-kB stimulation was observed.

[0135]

[0136] Table 3: Flow cytometry. Anti-4-1BB antibody binding to cell surface antigens.

[0137]

[0138] NF-κB reporter gene assay using Fc crosslinking from hFcgRIIA / CHOK1 cells and human 4-1BB 293 transfected cells

[0139] NF-κB Hu 4-1BB 293 transfected cells were transfected with 4-1BB in HEK-Dual TM TNF-α cells (Invivogen) were prepared and used to measure TNF-α-induced NF-kB activation. hFcgRIIA / CHOK1 transfected cells were used to provide cross-linking with anti-4-1BB IgG. They were taken up by Accutase, washed, and washed with DMEM (without phenol red) + 10% heat-inactivated fetal bovine serum at 1×10 6 Resuspend the cells at a rate of 5x10 cells / mL. 4NF-κB 4-1BB 293 transfected cells were co-cultured with the same number of hFcgRIIA / CHOK1 transfected cells or CHOK1 parental cells in Costar 3799 96-well U-bottom plates. Test antibodies, positive control antibodies (C1 and C2), and negative control antibodies (isotypes) were serially diluted in culture medium and added to the cells. After incubation at 37°C for 18 to 22 hours, 20 μl of cell suspension was removed from the wells and mixed with 50 μl of fluorescence assay reagent (Quanti-Luc, Invivo-Gen #rep-qlc) in an opaque 96 flat-bottom plate. Fluorescence (RLU) was measured on a Flexstation3 plate reader with an integration of 100 ms. ( Figure 1A and Figure 1B )(Table 4).

[0140] Table 4. NF-KB reporter gene activation induced by anti-4-1BB (anti-CD137) agonist antibodies.

[0141]

[0142] To detect 4-1BB-mediated T cell proliferation and interferon-γ release, T cells were prepared from peripheral blood mononuclear cells (PBMCs) using the Pan T cell isolation kit (Miltenyi Biotec product number 130-096-535) after transfection of CHOK1mFcgRIIB cells, resuspended in cold PBS, 0.5% bovine serum albumin (BSA) and 2mM EDTA, pH 7.2, and labeled with Cell Trace Violet (ThermoFisher, product number C34557) according to the kit instructions. Finally, the labeled pan T cells were isolated at 2x10 6 The cells were resuspended in Advanced RPMI-1640, 10% heat-inactivated FBS, 2-mercaptoethanol (1:1000) at a concentration of 10 cells / ml. CHOK1.mFcgRIIB-mCherry cells were cultured in F12 Ham, 10% FBS, 5 μg / ml puromycin, harvested by Accutase, and plated at 2x10 5The cells were resuspended in Advanced RPMI-1640, 10% heat-inactivated FBS, 2-mercaptoethanol (1:1000) at a concentration of 10 cells / ml. CHOK1 parental cells were prepared similarly, but without puromycin in the culture medium. 100,000 labeled Pan T cells / well were mixed with CHOK1mFcgRIIB-mCherry or CHOK1 at 10,000 cells / well in a 10:1 ratio in a 96-well U-bottom culture plate. 10 ng / ml NA / LE mouse anti-human CD3 (clone UCHT-1) was added to all wells, followed by serial dilutions of the 4-1BB test antibody, the positive control antibody (C1 or C2), and the negative control antibody (isotype) starting at a concentration of 1 μg / ml. Control wells included CHOK1mFcgRIIB-mCherry, Pan T cells + CHOK1mFcgRIIB, Pan T cells + CHOK1mFcgRIIB + anti-human CD3, Pan T cells + anti-human CD3, and Pan T cells. The plates were incubated at 37°C in a CO2 incubator for 5 days. Cells were further stained for flow cytometry with FITC anti-huCD3, PE anti-huCD8, and 7-AAD. Samples were analyzed using FlowJo by gating on FSC / SSC, followed by live / dead cell gating, followed by proliferation of CD3+ / CD8+ T cells and CD3+ / CD8- T cells (CellTrace Violet). Figure 2A and Figure 2B Anti-4-1BB (anti-CD137) agonist antibodies are shown to stimulate proliferation of primary CD3+CD8+ T cells. Figure 2C and Figure 2D Anti-4-1BB (anti-CD137) agonist antibodies are shown to stimulate proliferation of primary CD3+CD8- T cells.

[0143] The cytokine (IFN-γ) release assay is prepared similarly, but without pan T cell labeling. The plates are incubated at 37°C in a CO2 incubator for 48 hours, and the culture supernatant is separated and collected in two U-bottom 96-well plates (70-80 μl / plate). For Elisa, the culture supernatant is diluted 1:2 using reagent diluent (R&D Systems, Cat. No. DY995). Please refer to the R&D Systems data sheet and analytical procedure (Cat. No. DY285B). Figure 3A and Figure 3B Anti-4-1BB (anti-CD137) agonist antibodies are shown to stimulate interferon-γ secretion.

[0144] MC38 is growth inhibited in transgenic human 4-1BB knock-in mice

[0145] Mouse colon cancer MC38 cells were expanded in DMEM medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin and 2mM glutamine at 37°C in a 5% carbon dioxide incubator. 5E5 Mc38 cells were subcutaneously inoculated into the right axilla, and tumors grew to 50mm before the administration of the test article. 3 Up to 100mm 3 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of anti-4-1BB antibody or 3 mg / kg of hIgG2 were administered to 8 h4-1BB knock-in female mice per group twice a week for a total of 5 doses. Figure 4A It is shown that the body weight of all tested transgenic hu4-1BB knock-in mice gradually increased after administration of anti-4-1BB antibody. In addition, no drug-related toxicity was observed. QL1806 represents an anti-4-1BB antibody. Figure 4B It was shown that anti-4-1BB inhibited the growth of mouse colon cancer cells MC38. On day 21, significant inhibition (p<0.01) was observed, with 1 mg / kg and 3 mg / kg dose groups each containing two tumor-free mice (Table 5).

[0146] Table 5. Tumor volume, inhibition rate and tumor regression on day 21 (n=8)

[0147]

[0148] Note: *p<0.05; **p<0.01 compared with hIgG2 (3 mg / kg group).

[0149] The clone names (antibodies) shown in the Figures, Tables and Examples described herein include the heavy and light chain pairings shown in Table 6 below:

[0150] Table 6. Sequences of clone names

[0151] name Heavy chain SEQ ID NO Light chain SEQ ID NO G28.21 2 1 F1.1 36 35 huG28.21.G2.1.4 32 33 huG28.21.G2.4.4 34 33 huF1.1.G2.2.4 48 46 huF1.1.G2.4.4 50 46 QL1806 34 33

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

Claims

1. An isolated anti-CD137 agonist antibody or antigen-binding portion thereof, comprising: a heavy chain variable region CDR1 comprising SEQ ID NO: 6, a heavy chain variable region CDR2 comprising SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising SEQ ID NO: 8; and A light chain variable region CDR1 comprising SEQ ID NO: 3, a light chain variable region CDR2 comprising SEQ ID NO: 4; and a light chain variable region CDR3 comprising SEQ ID NO: 5; in, The antibody or antigen-binding portion thereof specifically binds to human CD137.

2. An isolated anti-CD137 agonist antibody or an antigen-binding portion thereof, comprising: a heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises SEQ ID NO: 29, a heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises SEQ ID NO: 30, and a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises SEQ ID NO:

31.

3. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof of claim 1 or 2, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

4. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof according to any one of claims 1 to 3, comprising a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO:

2.

5. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof according to any one of claims 1 to 4, which is a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single chain antibody or a bispecific antibody.

6. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof according to any one of claims 1 to 5, which is a chimeric antibody or a humanized antibody.

7. The isolated anti-CD137 agonist antibody or antigen binding portion thereof according to any one of claims 1 to 6, which is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule.

8. The isolated anti-CD137 agonist antibody or antigen binding portion thereof according to any one of claims 1 to 7, which is IgG1, IgG2, IgG3 or IgG4.

9. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof according to any one of claims 1 to 8, comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO:

12.

10. The isolated anti-CD137 agonist antibody or antigen binding portion thereof according to any one of claims 1 to 9, which is a chimeric antibody or a humanized antibody.

11. The isolated anti-CD137 agonist antibody or antigen binding portion thereof according to any one of claims 1 to 9, which is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule.

12. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof according to claim 10, which is IgG1, IgG2, IgG3 or IgG4.

13. The isolated anti-CD137 agonist antibody or antigen-binding portion thereof according to any one of claims 1 to 12, in, The antibody is a monoclonal antibody.

14. An isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:37 to SEQ ID NO:39; and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:40 to SEQ ID NO:

42.

15. A pharmaceutical composition comprising the isolated anti-CD137 agonist antibody or antigen binding portion thereof according to any one of claims 1 to 13, or the isolated monoclonal antibody or antigen binding portion thereof according to claim 14, and a pharmaceutically acceptable transporter.

16. Use of the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating cancer in a subject.

17. A method for evaluating the binding affinity of an antibody to human CD137, wherein include: The antibody is contacted with the recombinant CD137 protein, and the binding affinity is detected by a method selected from the group consisting of ELISA assay, flow cytometry assay, BIAcore binding assay, and OctetRed96.

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