Anti-CD137 antibodies and methods of making and using same

Abstract

Provided herein are masked anti-CD137 antibodies, nucleic acids encoding such antibodies, vectors comprising such nucleic acids, and host cells comprising such nucleic acids or vectors. Also provided are methods of producing the masked anti-CD137 antibodies. Further provided are methods of treating cancer comprising administering an effective amount of a masked anti-CD137 antibody as a monotherapy or in combination with an additional therapeutic antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority benefit of International Patent Application No. PCT / CN2022 / 079475 filed on March 7, 2022, the entire text of which is incorporated herein by reference.

[0003] References to Electronic Sequence Listings

[0004] The entire text of the electronic sequence listing (695402002241SEQLIST.xml; size: 126,181 bytes; and creation date: March 3, 2023) is incorporated herein by reference. Technical Field

[0005] The present disclosure relates to shielded anti-CD137 antibodies and antigen-binding fragments thereof that bind to human CD137, compositions comprising the same, and their use in delaying and / or preventing tumor growth. Background Art

[0006] CD137 (also known as CD137 receptor, 4-1BB, TNFRSF9, etc.) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). The current understanding of CD137 shows that its expression is usually activation-dependent and present in a wide range of subsets of immune cells, including activated NK and NKT cells, regulatory T cells, dendritic cells (DC), stimulated mast cells, differentiated bone marrow cells, monocytes, neutrophils and eosinophils (Wang, 2009, Immunological Reviews 229: 192-215). CD137 expression has been demonstrated to be present on tumor vasculature (Broll, 2001, Amer. J. Clin. Pathol. 115 (4): 543-549; Seaman, 2007, Cancer Cell 11: 539-554) and on endothelial sites of inflammation or atherosclerosis (Drenkard, 2007 FASEB J. 21: 456-463; Olofsson, 2008, Circulation 117: 1292-1301). The ligand that stimulates CD137, CD137 ligand (CD137L), is expressed on activated antigen presenting cells (APCs), bone marrow progenitor cells, and hematopoietic stem cells.

[0007] Human CD137 is a 255 amino acid protein (GenBank accession number NM_001561; NP_001552; SEQ ID NO.: 1). The protein comprises a signal sequence (amino acid residues 1-17), followed by an extracellular domain (169 amino acids), a transmembrane region (27 amino acids), and an intracellular domain (42 amino acids) (Cheuk ATC et al. 2004 Cancer Gene Therapy 11: 215-226). The receptor is expressed on the cell surface in monomeric and dimer forms and may trimerize with CD137 ligands for signal transduction.

[0008] Multiple studies of mouse and human T cells have shown that CD137 promotes enhanced cell proliferation, survival and cytokine production (Croft, 2009, Nat Rev Immunol 9: 271-285). Studies have shown that some CD137 agonist monoclonal antibodies (mAbs) increase the expression of co-stimulatory molecules and significantly enhance cytolytic T lymphocyte responses, thereby producing anti-tumor efficacy in various models. CD137 agonist mAbs have demonstrated efficacy in preventive and therapeutic settings. In addition, CD137 monotherapy and combination therapy tumor models have established persistent anti-tumor protective T cell memory responses (Lynch, 2008, Immunol Rev. 22: 277-286). CD137 agonists have also been shown to suppress autoimmune responses in a variety of art-recognized autoimmune models (Vinay, 2006, J Mol Med 84: 726-736). This dual activity of CD137 provides anti-tumor activity while preventing the potential for autoimmune side effects associated with immunotherapy that destroys immune tolerance.

[0009] However, liver-related autoimmune toxicity caused by agonistic anti-CD137 antibodies greatly limits their clinical application. There is a long-standing unmet need for antibodies that bind to human CD137, increase CD137-mediated responses, and provide for the treatment of various diseases and conditions (e.g., cancer) without immune-mediated side effects. Summary of the invention

[0010] In some embodiments, the shielded antibodies provided herein comprise a shielding peptide (MP) and an antibody that binds to human CD137, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL); wherein the MP is linked to the N-terminus of the VL, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus, and wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7; and wherein the VH comprises a CDR-H1 shown in TGGVGVG (SEQ ID NO: 36), a CDR-H2 shown in LIDWADDKYYSPSLKS (SEQ ID NO: 37), and a CDR-H3 shown in GGSDTVIGDWFAY (SEQ ID NO: 38), and wherein the VL comprises a CDR-L1 shown in RASQSIGSYLA (SEQ ID NO: 39), a CDR-L2 shown in DASNLET (SEQ ID NO: 40), and a CDR-L4 shown in QQGYYLWT (SEQ ID NO: 41). In some embodiments, the MP further comprises an N-terminal unit (NU) connected to the N-terminus of the MU. In some embodiments, the N-terminal unit is about 1-10 amino acid residues long. In some embodiments, the N-terminal unit comprises E or EVGSY (SEQ ID NO: 77).

[0011] In some embodiments, LU comprises a first cleavage site. In some embodiments, the first cleavage site is selected from the group consisting of protease cleavage sites of the following proteases: urokinase-type plasminogen activator / uPA, matrix metalloproteinase-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus protease / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Casparase-1, Casparase-2, Casparase-3, Casparase-4, Casparase-5, Casparase-6, Casparase-7, Casparase-8, Casparase-9, Casparase-10, Casparase-11, Casparase-12, Casparase-13, Casparase-14, and TACE. In some embodiments, the first cleavage site is a protease cleavage site of urokinase-type plasminogen activator / uPA or MMP-9. In some embodiments, LU further comprises a first linker (L1) to the C-terminus of the first cleavage site. In some embodiments, LU further comprises a second cleavage site. In some embodiments, the second cleavage site is at the C-terminus of L1. In some embodiments, the second cleavage site is a group consisting of protease cleavage sites of the following proteases: urokinase-type plasminogen activator / uPA, matrix metalloproteinase-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus protease / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, casperase-1, casperase-2, casperase-3, casperase-4, casperase-5, casperase-6, casperase-7, casperase-8, casperase-9, casperase-10, casperase-11, casperase-12, casperase-13, casperase-14 and TACE. In some embodiments, the second cleavage site is a protease cleavage site of urokinase-type plasminogen activator / uPA or MMP-9. In some embodiments, the first and second cleavage sites are the same. In some embodiments, the first and second cleavage sites are different. In some embodiments, LU further comprises a second linker (L2) to the C-terminal end of the second cleavage site. In some embodiments, LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-16.

[0012] In some embodiments, the shielding peptide comprises any one of SEQ ID NOs: 17-35. In some embodiments, the shielding peptide comprises SEQ ID NO: 34. In some embodiments, the antibody comprises the VH shown in SEQ ID NO: 52 and the VL shown in SEQ ID NO: 53. In some embodiments, the VH comprises SEQ ID NO: 52 and the VL comprises SEQ ID NO: 58.

[0013] In some embodiments, the masked antibody is a full-length antibody comprising an Fc region. In some embodiments, the Fc region is a human IgG Fc region or a variant thereof. In some embodiments, the human IgG Fc region or a variant thereof is a human IgG1 Fc region, a human IgG2 Fc region, a human IgG4 Fc region, or a variant of any of the foregoing. In some embodiments, the masked antibody comprises a protein selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R 30R;G237D, H268D, P238D and P271G;G237D, P238D, P271G and A330R;E233D, H268D, P238D, P271G and A330R;G237D, H268D, P238D, P271G and A330R;And E233D, G237D, P238D, H268D, P271G and A330R;S2657A;T437R;K248E;And T437R and K248E substitutions consisting of a group of human IgG1 Fc region variants, wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a human IgG1 Fc region variant containing S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises an Fc region comprising SEQ ID NO:113 or SEQ ID NO:114.

[0014] In some embodiments, the masked antibody comprises the masking peptide shown in SEQ ID NO: 34, the antibody heavy chain variable region shown in SEQ ID NO: 52 and the antibody light chain variable region shown in SEQ ID NO: 53, and a human IgG1 Fc region variant comprising S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO: 96. In some embodiments, the masked antibody comprises a protein selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R 30R;G237D, H268D, P238D and P271G;G237D, P238D, P271G and A330R;E233D, H268D, P238D, P271G and A330R;G237D, H268D, P238D, P271G and A330R;And E233D, G237D, P238D, H268D, P271G and A330R;S2657A;T437R;K248E;And T437R and K248E substitutions consisting of a group of human IgG4 Fc region variants, wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a human IgG4 Fc region variant containing S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises an Fc region comprising SEQ ID NO: 117 or 118. In some embodiments, the masked antibody comprises a masking peptide of SEQ ID NO: 34, an antibody heavy chain variable region as shown in SEQ ID NO: 52 and an antibody light chain variable region as shown in SEQ ID NO: 53, and a human IgG4 Fc region variant comprising S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a heavy chain comprising SEQ ID NO: 92 or 93 and a light chain comprising SEQ ID NO: 96.

[0015] In some embodiments, the masked antibody is a masked antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, (scFv)2, linear antibody, single-chain antibody, single-domain antibody (nanobody) VHH, minibody or diabody.

[0016] In some embodiments, one or more polynucleotides encoding the masked antibodies described herein are provided. In some embodiments, a recombinant vector comprising one or more polynucleotides described herein is provided. In some embodiments, a host cell comprising the vector described herein is provided. In some embodiments, a method of producing a masked antibody is provided, comprising culturing the host cell in the claims described herein under appropriate conditions to cause expression of the masked antibody and recovering the masked antibody.

[0017] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of a shielded antibody provided herein. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor is breast cancer, liver cancer, colorectal cancer, or colon cancer.

[0018] In some embodiments, a method for treating cancer in a subject is provided, comprising administering to the subject an effective amount of a shielded antibody as described herein and an effective amount of an anti-PD-1 antibody (e.g., an anti-human PD-1 antibody). In some embodiments, a method for treating cancer in a subject is provided, comprising administering to the subject an effective amount of a shielded antibody as described herein and an effective amount of an anti-CTLA4 antibody (e.g., an anti-human CTLA4 antibody). In some embodiments, a method for treating cancer in a subject is provided, comprising administering to the subject an effective amount of a shielded antibody as described herein and an effective amount of a bispecific antibody that binds HER2 (e.g., human HER2) and CD3 (e.g., human CD3). In some embodiments, a method for treating cancer in a subject is provided, comprising administering to the subject an effective amount of a shielded antibody as described herein and an effective amount of a bispecific T cell engager (TCE) targeting CD3 (e.g., human CD3) and an antigen expressed on the surface of a solid tumor cancer cell. In some embodiments, the antigen expressed on the surface of a solid tumor is HER2 (e.g., human HER2). In some embodiments, the TCE targeting CD3 and HER2 comprises three polypeptide chains, wherein the first polypeptide chain comprises an antibody heavy chain, the second polypeptide chain comprises an antibody light chain, and the third polypeptide chain comprises scFv-Fc, wherein the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, wherein the first and second polypeptide chains associate to form a HER-2 binding arm, wherein the third polypeptide chain binds CD3, and wherein the Fc domain of the anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form a TCE. In some embodiments, the TCE is a shielded TCE (e.g., wherein the portion of the TCE binding to CD3 and / or the portion of the TCE binding to HER2 is shielded). In some embodiments, the first polypeptide chain comprises SEQ ID NO: 125, the second polypeptide chain comprises SEQ ID NO: 124, and the third polypeptide chain comprises SEQ ID NO: 126. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is colon cancer, breast cancer, liver cancer, colorectal cancer, or colon cancer.

[0019] In some embodiments, a kit comprising a masked antibody as described herein is provided, which is used to treat an individual with cancer according to the methods described herein. In some embodiments, a kit comprising a masked antibody as described herein is provided, which is used in combination with an anti-PD-1 antibody (e.g., an anti-human PD-1 antibody) to treat an individual with cancer according to the methods described herein. In some embodiments, a kit comprising a masked antibody as described herein is provided, which is used in combination with an anti-CTLA4 antibody (e.g., an anti-human CTLA4 antibody) to treat an individual with cancer according to the methods described herein. In some embodiments, a kit comprising a masked antibody as described herein is provided, which is used in combination with a bispecific antibody that binds HER2 (e.g., human HER2) and CD3 (e.g., human CD3) to treat an individual with cancer according to the methods described herein. In some embodiments, a kit comprising a masked antibody as described herein is provided, which is used in combination with a bispecific T cell engager (TCE) targeting CD3 (e.g., human CD3) and an antigen expressed on the surface of a solid tumor cancer cell to treat an individual with cancer according to the methods described herein. In some embodiments, the antigen expressed on the surface of a solid tumor is HER2 (e.g., human HER2). In some embodiments, the TCE targeting CD3 and HER2 comprises three polypeptide chains, wherein the first polypeptide chain comprises an antibody heavy chain, the second polypeptide chain comprises an antibody light chain, and the third polypeptide chain comprises scFv-Fc, wherein the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, wherein the first and second polypeptide chains associate to form a HER-2 binding arm, wherein the third polypeptide chain binds CD3, and wherein the Fc domain of the anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form a TCE. In some embodiments, the TCE is a shielded TCE (e.g., wherein the portion of the TCE binding to CD3 and / or the portion of the TCE binding to HER2 is shielded). In some embodiments, the first polypeptide chain comprises SEQ ID NO: 125, the second polypeptide chain comprises SEQ ID NO: 124, and the third polypeptide chain comprises SEQ ID NO: 126. It should be understood that one, some or all of the properties of the various embodiments described above and herein can be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A Results of a FACS-based assay performed to evaluate the masking efficiency of exemplary CD137 masked antibodies in displaying human CD137 on the surface of yeast cells are shown and compared to parental antibodies TY21242 and TY23310. Figure 1BAlso shown are the results of a FACS-based assay performed to assess the masking efficiency of exemplary CD137 masked antibodies in displaying human CD137 on the surface of yeast cells and compared to the parental antibodies TY21242 and TY23310. Figure 1C Additional results are shown for a FACS-based assay performed to evaluate the masking efficiency of exemplary CD137 masked antibodies in displaying human CD137 on the surface of yeast cells and compared to parental antibodies TY21242 and TY23310. Figure 1D Further results are shown for a FACS-based assay evaluating the masking efficiency of exemplary CD137 masked antibodies in displaying human CD137 on the surface of yeast cells and compared to the parental antibodies TY21242 and TY23310.

[0021] Figure 2A The results of ELISA assays performed to assess the activity of masked anti-CD137 antibodies TY25366 and TY25368 compared to parental antibodies TY24118 and TY24122, respectively, are shown before removal of the masking peptide (ie, without MMP9 treatment) and after removal of the masking peptide (ie, with MMP9 treatment). See also Table D.

[0022] Figure 2B The results of a FACS-based assay performed to assess the activity of masked anti-CD137 antibodies TY25366 and TY25368 compared to parental antibodies TY24118 and TY24122, respectively, are shown before removal of the masking peptide (ie, without MMP9 treatment) and after removal of the masking peptide (ie, with MMP9 treatment). See also Table D.

[0023] Figures 3A to 3E show size exclusion chromatography (SEC) profiles of exemplary masked antibodies TY25366 and TY25368 under accelerated stress conditions. Figure 3A (i) shows the SEC profiles of TY25366 after 0, 3, and 6 freeze and thaw cycles. Figure 3A (ii) shows the SEC profiles of TY25368 after 0, 3, and 6 freeze and thaw cycles. Figure 3B (i) shows the SEC profiles of TY25366 after storage at 40°C for 0, 7, 14, 21, and 28 days. Figure 3B (ii) shows the SEC profiles of TY25368 after storage at 40°C for 0, 7, 14, 21, and 28 days. Figure 3C (i) shows the SEC profiles of TY25366 after storage at room temperature for 0 and 2 hours in an acidic buffer (sodium acetate solution, pH 3.6). Figure 3C (ii) shows the SEC profiles of TY25368 after storage at room temperature for 0 and 2 hours in the same acidic buffer. Figure 3D(i) shows the SEC profile of TY25366 after storage in 50 mM histidine, 300 mM NaCl, pH 7.0 buffer at 40°C for 0 and 24 hours. Figure 3D(ii) shows the SEC profile of TY25368 after storage in 50 mM histidine, 300 mM NaCl, pH 7.0 buffer at 40°C for 0 and 24 hours. Figure 3E(i) shows the SEC profiles of TY25366 and TY25368 before and after storage in saline for 6 hours and at room temperature at 4°C for 24 hours compared to the control condition.

[0024] FIG4A(i) shows the evaluation of the interaction between TY25368 treated with and without MMP9 and naive and activated CD4 + Figure 4A(ii) shows the results of a FACS-based assay to assess the binding of TY25368 to naive and activated CD8 + Figure 4B (i) shows the results of a FACS-based assay to assess the binding of TY25368 to naive and activated CD4 + Figure 4B (ii) shows the results of a FACS-based assay to assess the binding of TY25368 to naive and activated CD8 + Figure 4C (i) shows the results of a FACS-based assay to assess the binding of TY25368 to naive and activated CD4 + Figure 4C (ii) shows the results of a FACS-based assay to assess the binding of TY25368 to naive and activated CD8 +Figure 4D (i) shows the results of a FACS-based assay to assess the binding of TY25368 to naive and activated CD4 + Figure 4D (ii) shows the results of a FACS-based assay to assess the binding of MMP9-treated and untreated TY25368 to naive and activated CD8 + Results of a FACS-based assay of rat T cell binding.

[0025] Figure 5 Shown are the results of an ELISA experiment performed to assess the extent to which MMP9-treated and untreated TY25368 blocks the binding of CD137 to its ligand.

[0026] Fig. 6A Shown are the results of experiments performed to assess the extent to which different masked and unmasked anti-CD137 antibodies stimulate CD137 signaling.CD137-mediated signaling was measured using a CD137 reporter gene assay in the presence of CHO-K1-hFcgRIIb cells as a cross-linking agent (E:CL=20:1). Figure 6B Shown are the results of experiments performed to assess the extent to which different blocked and unblocked anti-CD137 antibodies stimulate CD137 signaling.CD137-mediated signaling was measured using a CD137 reporter gene assay in the absence of CHO-K1-hFcgRIIb cells as a cross-linking agent.

[0027] Fig. 7A Shown are the results of experiments performed to assess the extent to which different blocked and unblocked anti-CD137 antibodies stimulate CD137 signaling.CD137-mediated signaling was measured using a CD137 reporter gene assay in the presence of human primary B cells as a cross-linking agent (E:CL=5:1). Figure 7B Shown are the results of experiments performed to assess the extent to which different blocked and unblocked anti-CD137 antibodies stimulate CD137 signaling.CD137-mediated signaling was measured using a CD137 reporter gene assay in the presence of human primary B cells as a cross-linking agent (E:CL=20:1).

[0028] Fig. 8A Shown are the results of an ELISA-based assay performed to determine whether anti-CD137 antibodies enhance SEA-stimulated cytokine secretion from human PBMCs obtained from donor #102. Figure 8B Shown are the results of an ELISA-based assay performed to determine whether anti-CD137 antibodies enhance SEA-stimulated cytokine secretion from human PBMCs obtained from donor #142.

[0029] Fig. 9Shown are the results of experiments performed to evaluate the ADCC activity of anti-CD137 antibodies and masked anti-CD137 antibodies.

[0030] Fig.10 Shown are the results of experiments performed to evaluate the CDC activity of masked anti-CD137 antibodies.

[0031] Fig.11 Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) masked anti-CD137 antibody TY25368, (c) anti-PD-1 antibody FG1225, and (d) TY25368 and FG1225 in combination in inhibiting the growth of CT26 murine colon tumors in a mouse allograft model.

[0032] Fig.12 Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) shielded anti-CD137 antibody TY25368, (c) unshielded anti-CTLA4 antibody TY21580, (d) shielded anti-CTLA4 antibody TY22404, (e) shielded anti-CD137 antibody TY25368 and unshielded anti-CTLA4 antibody TY21580, and (f) shielded anti-CD137 antibody TY25368 and shielded anti-CTLA4 antibody TY22404 in inhibiting the growth of MC28 murine colon tumors in a mouse allograft model.

[0033] Fig.13 Provided are concentration-time profiles of intact and total forms of TY25366 and TY25368 at doses of 30 and 100 mg / kg in cynomolgus monkeys.

[0034] Fig.14A Shown are the results of experiments performed to evaluate the activation of CD137-mediated cell signaling by anti-CD137 antibodies containing Fc mutations in the presence of CHO-K1-hFcγRIIb as a cross-linking agent. Fig. 14B Shown are the results of experiments conducted to evaluate the activation of CD137-mediated cell signaling by anti-CD137 antibodies containing Fc mutations in the presence of CHO-K1-mFcγRIIb as a cross-linking agent. Fig. 14C Shown are the results of experiments performed to assess the activation of CD137-mediated cell signaling by anti-CD137 antibodies comprising Fc mutations in the absence of cross-linking agents.

[0035] Fig.15A Shown are the results of experiments performed to evaluate the extent to which anti-CD137 antibodies containing Fc mutations immobilized on a solid support enhance SEA-stimulated cytokine secretion from human PBMCs. Fig. 15BShown are the results of experiments performed to evaluate the extent to which soluble anti-CD137 antibodies containing Fc mutations enhance SEA-stimulated cytokine secretion from human PBMCs.

[0036] Fig.16 Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) 3 mg / kg of shielded anti-CD137 antibody TY25368, (c) 1 mg / kg of shielded anti-CD137 antibody TY25368, and (d) 0.3 mg / kg of shielded anti-CD137 antibody TY25368 in inhibiting the growth of EMT6 murine breast cancer tumors in a mouse allograft model.

[0037] Fig.17 Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) 5 mg / kg TY24118, (c) 5 mg / kg TY24122, (d) 5 mg / kg TY25366, or (e) 5 mg / kg TY25368 in inhibiting H22 murine hepatocarcinoma tumor growth in a mouse allograft model.

[0038] Fig.18A Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) 5 mg / kg TY24118, (c) 5 mg / kg TY24122, (d) 5 mg / kg TY25366, or (e) 5 mg / kg TY25368 in inhibiting the growth of CT26 murine colorectal carcinoma tumors in a mouse allograft model. Fig.18B Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) 1 mg / kg TY24118, (c) 1 mg / kg TY24122, (d) 1 mg / kg TY25366, or (e) 1 mg / kg TY25368 in inhibiting the growth of CT26 murine colorectal carcinoma tumors in a mouse allograft model.

[0039] Fig.19A Provided are the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) 5 mg / kg TY25368, (c) 5 mg / kg TY27151, or (d) 5 mg / kg TY25368 and 5 mg / kg TY27151 in inhibiting the growth of MC38 murine colon carcinoma tumors expressing human HER2 and human B7H3 in a mouse syngeneic tumor model. Fig.19B Tumor growth curves for individual mice in each treatment group (a), (b), (c), and (d) are provided.

[0040] Fig. 20 A schematic description of TY27151 is provided. DETAILED DESCRIPTION

[0041] Overview

[0042] The toxicity of widely stimulated T cells limits the applicability of many CD137 agonists in cancer immunotherapy. The shielded anti-CD137 antibodies provided by the present disclosure can effectively treat cancer without causing major safety issues.

[0043] definition

[0044] Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0045] As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more of those molecules, and so on.

[0046] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments related to that value or parameter itself.

[0047] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0048] As used herein, the term "and / or", phrases such as "A and / or B" are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or", phrases such as "A, B and / or C" are intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0049] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

[0050] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to nucleotide polymers of any length, and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interspersed with non-nucleotide components. Polynucleotides may contain one or more modifications performed after synthesis, such as conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomer nucleic acids, etc.), and unmodified forms of one or more polynucleotides. In addition, any hydroxyl group normally present in a sugar may be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with additional nucleotides, or may be conjugated to a solid or semisolid support. The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping groups having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to obtain standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose that are generally known in the art, including, for example, 2'-O-methyl-ribose; 2'-O-allyl-ribose; 2'-fluoro-ribose or 2'-azido-ribose; carbocyclic sugar analogs; α-anomeric sugars; epimeric sugars such as arabinose, xylose or lyxose; pyranose; furanose; sedoheptulose; acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("methylal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, an aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in a polynucleotide need be identical.The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0051] The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA or synthetic origin, or a combination thereof, that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from chromosomes that are naturally associated with the genomic DNA. Preferably, an "isolated" nucleic acid does not contain sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the target nucleic acid).

[0052] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masked antibodies (e.g., activatable or non-activatable antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., single-chain variable fragments or scFvs) so long as they exhibit the desired biological activity (e.g., the ability to bind to a target antigen with the desired specificity and affinity).

[0053] In some embodiments, the term "antibody" refers to an antigen binding protein (i.e., immunoglobulin) having a basic four polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as V H ), followed by the constant region. The heavy chain constant region consists of three domains: C H1 , C H2 and C H3 Each light chain has a variable region (abbreviated herein as V I ), followed by a constant region at the other end. The light chain constant region consists of one domain, C L . V L With V H Compare and C L Aligned with the first constant domain (CH1) of the heavy chain. H and V L The IgM antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, thus containing 10 antigen binding sites; while secretory IgA antibodies can polymerize to form multivalent assemblies containing 2-5 basic 4-chain units and J chain.

[0054] V H and V LThe V region can also be further subdivided based on structure and sequence analysis into regions of high variability, termed hypervariable regions (HVRs). HVRs are interspersed with more conserved regions, termed framework regions (FWs) (see, e.g., Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V H and V L It consists of three HVRs and four FWs, arranged from the amino terminus to the carboxyl terminus in the following order: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs of the light chain are referred to as HVR-L1, HVR-L2, and HVR-L3.

[0055] Table 1 below provides exemplary CDR definitions according to various algorithms known in the art.

[0056] Table 1. CDR definitions

[0057] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> <![CDATA[MacCallum 3 ]]> <![CDATA[IMGT 4 ]]> <![CDATA[AHo 5 ]]> CDR-H1 31-35 26-32 30-35 27-38 25-40 CDR-H2 50-65 53-55 47-58 56-65 58-77 CDR-H3 95-102 96-101 93-101 105-117 109-137 CDR-L1 24-34 26-32 30-36 27-38 25-40 CDR-L2 50-56 50-52 46-55 56-65 58-77 CDR-L3 89-97 91-96 89-96 105-117 109-137

[0058] 1 The residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U. Sept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991).

[0059] 2 The residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997).

[0060] 3 Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008).

[0061] 4Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).

[0062] 5 Residue numbering follows the nomenclature of Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).

[0063] The variable region of heavy chain and light chain contains the binding domain that interacts with antigen.The constant region of antibody can mediate the combination of immunoglobulin and host tissue or factor, including various cells (e.g., effector cells) of immune system and the first component (C1q) of classical complement system.In light chain and heavy chain, variable region and constant region are connected by " J " district with about 12 or more amino acid, and heavy chain also includes " D " district with about 10 or more amino acid (referring to, for example, Fundamental Immunology the 7th chapter (Paul, W. compiles, the 2nd edition, Raven Press, NY). (1989)).

[0064] The L chain from any vertebrate species can be designated as one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain. Antibodies can be designated as different species or isotypes depending on the amino acid sequence of the constant domain (CH) of its heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. IgG class antibodies can be further classified into four subclasses IgG1, IgG2, IgG3, and IgG4 by γ heavy chains Y1-Y4, respectively.

[0065] The terms "antigen-binding fragment", "antigen-binding portion" or "antigen-binding domain" of an antibody are used interchangeably and refer to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds. Examples of "antigen-binding fragments" of antibodies include, but are not limited to, (i) a Fab fragment, which is a fragment consisting of 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 a disulfide bond in the hinge region; and (iii) a V fragment consisting of a single arm of an antibody. L and V HFv fragments composed of domains. Other exemplary antigen-binding fragments are described elsewhere herein.

[0066] The term "shielded antibody" refers to an antibody or antigen-binding fragment thereof comprising a shielding peptide that interferes, hinders, reduces (ability), prevents, inhibits binding to its target, or competes with the antigen-binding domain of the antibody for binding to its target. Shielded antibodies can be produced by connecting a shielding peptide to the antigen-binding domain of an antibody. In some embodiments, a shielded antibody or antigen-binding fragment thereof exhibits a first binding affinity to a target in an unactivated state (e.g., inhibited or shielded by a shielding peptide), and exhibits a second binding affinity to a target in an activated state (e.g., not inhibited or shielded by a shielding peptide (e.g., a shielding peptide cleaved from an antibody)), wherein the second binding affinity is greater than the first binding affinity. Shielded antibodies can be produced by connecting a shielding peptide comprising an activatable portion (e.g., a cleavable site or "LU" of a linking unit) to the antigen-binding domain of an antibody.

[0067] The term "shielding peptide" refers to a peptide that inhibits the binding of an antigen-binding domain to its target antigen, and generally comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus. The C-terminus of the shielding peptide is generally connected to the N-terminus of the VH or VL of the antigen-binding domain. In some embodiments, the shielding peptide or a portion thereof interferes with or inhibits the binding of the antigen-binding domain to its target so effectively that the antigen-binding domain binds to its target extremely low and / or below the detection limit (e.g., undetectable binding in ELISA or flow cytometry). The shielded antibody or polypeptide used herein may comprise one or more connectors, for example, within the LU, between the MU and the LU, between the LU and the VH or VL, or between the VH and the Fc hinge region.

[0068] The LU of the shielding peptide may include at least one cleavable site. The cleavage site generally includes a cleavable amino acid sequence, for example, used as a substrate for an enzyme and / or cysteine-cysteine ​​pair that can form a reducible disulfide bond. Thus, when the terms "cleavage", "cleavable", "cleaved" and the like are used in conjunction with the cleavage site, those terms encompass, for example, enzymatic cleavage by a protease, and the interruption of the reduction of a disulfide bond between a cysteine-cysteine ​​pair by a disulfide bond that can be generated by exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with the MU or be contained within the MU. The shielded antibody or shielded polypeptide may include a cleavage site configured to mediate antibody or polypeptide activation. For example, when the cleavage site of the activatable antibody is intact (e.g., not cleaved by the corresponding enzyme, and / or containing an unreduced cysteine-cysteine ​​disulfide bond), the shielding peptide or a portion thereof may interfere with or inhibit the antigen-binding domain from binding to its target. In some embodiments, the LU of the shielding peptide does not include a cleavable site.

[0069] The term "shielding efficiency" refers to the efficiency of a shielding peptide in inhibiting the binding of an antigen binding domain to a target antigen. The shielding efficiency can be measured by the difference or ratio of the binding affinity of a shielded antibody or a shielded polypeptide comprising an antigen binding domain relative to the binding affinity of an unshielded antibody or an unshielded polypeptide comprising an antigen binding domain (e.g., a shielding peptide cleaved from an antibody). For example, the shielding efficiency can be measured by the EC50 or K of the inactive state of the shielded antibody (e.g., inhibiting, shielded and / or uncleaved) binding to the target antigen. D divided by the EC50 or K of the activated state of the unmasked antibody (e.g., uninhibited, unmasked, and / or cleaved) binding to the target antigen. D , or the EC50 or K of the parent antibody (e.g., not linked to a shielding peptide) binding to the target antigen D EC50 values ​​can be measured by ELISA measurement or Jurkat NFAT reporter analysis, such as described in US Patent Publication No. US20210207126A1. D The value can be measured by, for example, using surface plasmon resonance.

[0070] The term "epitope" refers to the part of an antigen that is combined with an antibody (or its antigen-binding fragment). An epitope can be formed by continuous or discontinuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by continuous amino acids are usually retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are usually lost when treated with a denaturing solvent. An epitope can include different numbers of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with antigens and computer modeling of their complex structures with binding antibodies and variants thereof. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by G.E. Morris (1996). Once the desired epitope of an antigen is determined, antibodies against the epitope can be generated, for example, using the techniques described herein. The generation and characterization of antibodies can also clarify information about the desired epitope. With this information, antibodies that bind to the same epitope can then be competitively screened. A way to achieve this is to perform cross-competition studies to find antibodies that compete with each other for binding, i.e., antibodies that compete for binding to the antigen. A high throughput method for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0071] The term "germline" refers to the nucleotide sequences of antibody genes and gene fragments as they are passed from parents to offspring through germ cells. Germline sequences differ from nucleotide sequences encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during B cell maturation.

[0072] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a sugar residue attachment position. The amino acids to which carbohydrates (e.g., oligosaccharides) are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. A specific attachment site is typically represented by an amino acid sequence, referred to herein as a "glycosylation site sequence". The glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to chemical groups that act as attachment sites between sugar molecules and amino acid residues. N-linked sugars are linked via amino groups; O-linked sugars are linked via hydroxyl groups. The term "glycan occupancy" refers to the presence of a carbohydrate moiety connected to a glycosylation site (i.e., the glycan site is occupied). In the case of at least two potential glycosylation sites on a polypeptide, the carbohydrate moiety may not be occupied (0-glycan site occupied), one site occupied (1-glycan site occupied), or two sites (2-glycan site occupied).

[0073] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include but are not limited to cultured cells, such as mammalian cultured cells such as CHO, BHK, NSO, SP2 / 0, YB2 / 0 derived from rodents (rat, mouse, guinea pig or hamster); human cells, such as HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (such as S2 cells), bacterial cells (such as Escherichia coli (E. coli) cells) and cells contained in transgenic animals or cultured tissues. The term not only covers specific subject cells, but also covers the progeny of such cells. Because some modifications may occur in succession due to mutations or environmental influences, the progeny may not be the same as the parent cell, but is still included in the scope of the term "host cell".

[0074] A "human antibody" is an antibody having an amino acid sequence corresponding to that produced by a human or human cell or derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0075] The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues derived from human antibody sequences. A humanized antibody may comprise some or all CDRs or HVRs from non-human animals or synthetic antibodies, while the framework and constant regions of the antibody comprise amino acid residues derived from human antibody sequences.

[0076] The term "exemplary antibody" refers to any of the antibodies described herein. These antibodies can be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above encompasses antibodies with the same V L Area and V H In addition, antibodies in the IgG category may be in any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each of the antibodies identified above that are in the IgG subclass encompass antibodies in all four subclasses that have the same V L Area and V H The amino acid sequences of the heavy chain constant regions of human antibodies in five classes and in four IgG subclasses are known in the art. The present disclosure provides the amino acid sequences of the full-length heavy and light chains of the IgG4 subclass of each illustrative antibody shown in Table 1b.

[0077] An "isolated" antibody or binding molecule is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0078] The term “k a ” refers to the association rate constant for a specific antibody-antigen interaction, while the term “k d ” refers to the dissociation rate constant for a specific antibody-antigen interaction.

[0079] The term "K D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. It is represented by k d With k a The ratio (i.e., k d / k a ) and expressed as molar concentration (M). K D Used as a measure of the affinity of the binding of an antibody to its binding partner. D The smaller the K, the tighter the antibody binds, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind more tightly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. D The K value can be determined using methods well established in the art. DA method is by using ELISA. For example, a detection procedure using ELISA.

[0080] The term "mammal" refers to any animal species of the class Mammalia. Examples of mammals include: humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domestic animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, etc.

[0081] The terms "prevent" or "preventing" with respect to a disease condition in a mammal means preventing or delaying the onset of the disease, or preventing the appearance of clinical or subclinical symptoms thereof.

[0082] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. The amino acid sequence identity of a polypeptide can be routinely determined using known computer programs such as Bestfit, FASTA or BLAST (see, e.g., Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucelic Acids Res. 25: 3389-3402 (1997)). When Bestfit or any other sequence alignment program is used to determine whether a particular sequence has, for example, 95% identity with a reference amino acid sequence, parameters are set so that the percentage of identity is calculated over the full length of the reference amino acid sequence and the interval of homology of the total number of amino acid residues in the reference sequence is allowed to be up to 5%. This above-described method of determining percent identity between polypeptides is applicable to all proteins, fragments or variants thereof disclosed herein.

[0083] As used herein, the terms "bind", "specifically bind", or "specific for" refer to a measurable and reproducible interaction, such as binding, between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. For example, a masked anti-CD137 antibody described herein is said to selectively bind to human CD137 if its EC50 for binding to human CD137 is less than 10% of its EC50 for binding to a different antigen as in an in vitro assay.

[0084] The term "treat, treating or treatment" with respect to a disease condition in a mammal refers to causing a desired or beneficial effect in a mammal suffering from the disease condition. The desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells, etc.), or a stop or inhibition of further development of the disease, condition or illness. In the context of treating cancer in a mammal, the desired or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or improvement in mammal survival. The effect may be subjective or objective. For example, if the mammal is a human, then the human may record improved energy or vitality or reduced pain as improved subjective symptoms or responses to therapy. Alternatively, a clinician may notice a reduction in tumor size or tumor load based on physical examination, laboratory parameters, tumor markers or imaging findings. For response to treatment, some laboratory signs that clinicians can observe include standardization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, clinicians can observe a reduction in detectable tumor markers. Alternatively, other tests can be used to assess objective improvement, such as sonograms, nuclear magnetic resonance tests, and positron emission tests.

[0085] The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. Foreign nucleic acid molecules are connected to carrier nucleic acid molecules by recombinant techniques such as connection or recombination. This allows breeding, selection, further manipulation and / or expression of foreign nucleic acid molecules in host cells or organisms. The vector can be a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus or a virion. One type of vector can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome (e.g., non-episomal mammalian vectors). Another type of vector can replicate autonomously in the host cell it introduces (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Another specific type of vector capable of directing the expression of the expressible foreign nucleic acid to which they are operatively connected is generally referred to as an "expression vector". An expression vector generally has a control sequence that drives the expression of the expressible foreign nucleic acid. The simpler vectors referred to as "transcription vectors" can only be transcribed but not translated: they can be replicated but not expressed in target cells. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of directing the expression of expressible nucleic acids to which they are operably linked are generally referred to as "expression vectors." Other examples of "vectors" may include display vectors (e.g., vectors that direct the expression and display of encoded polypeptides on the surface of viruses or cells (such as bacterial cells, yeast cells, insect cells, and / or mammalian cells)).

[0086] As used herein, "subject", "patient" or "individual" may refer to a human or a non-human animal. "Non-human animal" may refer to any animal that is not classified as a human, such as domestic animals, farm animals or zoo animals, sports animals, pet animals (such as dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer to nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.) without limitation. In some embodiments, the subject, patient or individual is a human.

[0087] "Effective amount" refers to at least the following quantity: at the necessary dosage and for the necessary period, the quantity effectively achieves one or more desired or indicated effects including treatment or prevention results. The effective amount can be provided by one or more administrations. For the purposes of this disclosure, the effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As understood in clinical situations, the effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., an effective amount as administered in the form of a monotherapy or combination therapy). Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and if in conjunction with one or more other agents, a desirable result can be achieved or achieved, then a single agent can be considered to be given in an effective amount.

[0088] Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to methods described in various general and more specific references well known in the art and as cited and discussed throughout this specification. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). Enzymatic reactions and purification techniques are performed according to manufacturer specifications, as generally achieved in the art or as described herein. The nomenclature used in combination with analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein, and the laboratory procedures and techniques of the analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry are those well known in the art and commonly used. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and the treatment of patients.

[0089] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0090] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0091] Masked anti-CD137 antibody

[0092] In one aspect, a shielded antibody is provided comprising a shielding peptide (MP) and an antibody that binds to human CD137 (hCD137), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the MP is linked to the N-terminus of the VL, wherein the MP comprises a shielding unit (MU), a linking unit (LU) from the N-terminus to the C-terminus, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, and wherein the VH comprises a CDR-H1 shown in TGGVGVG (SEQ ID NO: 36), a CDR-H2 shown in LIDWADDKYYSPSLKS (SEQ ID NO: 37), and a CDR-H3 shown in GGSDTVIGDWFAY (SEQ ID NO: 38), and wherein the VL comprises a CDR-L1 shown in RASQSIGSYLA (SEQ ID NO: 39), a CDR-L2 shown in DASNLET (SEQ ID NO: 40), and a CDR-L4 shown in QQGYYLWT (SEQ ID NO: 41). NO:41). In some embodiments, a shielded antibody is provided that comprises a shielding peptide (MP) and an antibody that binds to human CD137 (hCD137), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the MP is linked to the N-terminus of the VL, wherein the MP comprises, from the N-terminus to the C-terminus, a shielding unit (MU), a linking unit (LU), and wherein the VH comprises the CDR-H1 shown in TSGVGVG (SEQ ID NO:42), the CDR-H2 shown in LIDWDDDKYYSPSLKS (SEQ ID NO:43), and the CDR-H3 shown in GGSDTVLGDWFAY (SEQ ID NO:44), and wherein the VL comprises the CDR-L1 shown in RASQSVSPYLA (SEQ ID NO:45), the CDR-L2 shown in DASSLES (SEQ ID NO:46), and the CDR-L3 shown in QQGYSLWT (SEQ ID NO:47).In some embodiments, a shielded antibody is provided that comprises a shielding peptide (MP) and an antibody that binds to human CD137 (hCD137), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the MP is linked to the N-terminus of the VL, wherein the MP comprises a shielding unit (MU), a linking unit (LU) from the N-terminus to the C-terminus, and wherein the VH comprises the CDR-H1 shown in SGHYWA (SEQ ID NO:48), the CDR-H2 shown in SISGYGSTTYYADSVKG (SEQ ID NO:49), and the CDR-H3 shown in GGSDAVLGDWFAY (SEQ ID NO:50), and wherein the VL comprises the CDR-L1 shown in RASQGIGSFLA (SEQ ID NO:51), the CDR-L2 shown in DASNLET (SEQ ID NO:40), and the CDR-L3 shown in QQGYYLWT (SEQ ID NO:41). SEQ ID NOs: 1-7 are provided in Table 2 below:.

[0093] Table 2. Exemplary shielding unit sequences (SEQ ID NOs: 1-7)

[0094] SEQ ID NO: MU sequence 1 PTDLDACADAPNHCHF 2 SSTHAHCHHSPANCIS 3 DTDYDFCPILRHRCDS 4 NDYNYHCKWRPSRCHN 5 YHDYDDCRVLPRRCFN 6 SNNFASCLWRHRSCAD 7 TDNYDYCPRLRRKCYH

[0095] In some embodiments, MP further comprises an N-terminal unit. In some embodiments, the N-terminal unit length is between about 1 and 10 amino acids. In some embodiments, the N-terminal unit comprises E (glutamic acid) or EVGSY (SEQ ID NO: 77). In some embodiments, LU comprises at least one first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, LU further comprises a second cleavage site (CS2). In some embodiments, the first and / or second cleavage site is a protease cleavage site. In some embodiments, the first and second cleavage sites are the same. In some embodiments, the first and second cleavage sites are different. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to co-localize with a target of an activatable antibody comprising a cleavage site) can be used, including, for example, protease cleavage sites that are recognized and / or cleaved by urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP- 13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26 and / or MMP-27); tobacco etch virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMT S1, ADAMTS4 and / or ADAMTS5); caspers (e.g., caspers-1, caspers-2, caspers-3, caspers-4, caspers-5, caspers-6, caspers-7, caspers-8, caspers-9, caspers-10, caspers-11, caspers-12, caspers-13 and / or caspers-14); aspartic proteases (e.g., RACE and / or Renin); aspartic cathepsins (e.g., cathepsin D and / or cathepsin E); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2 and / or cathepsin X / Z / P); cysteine ​​proteases (e.g., Cruzipain, Legumain and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14);Metalloproteinases (e.g., transmembrane peptidase (Meprin), neprilysin (Neprilysin), PSMA and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase (Chymase) and / or coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granzyme B; guanidinobenzoic acid enzymes; HtrA1; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine protease (TTSP) (e.g., DESC1, DPP-4, FAP, hepsin, matrix proteinase-2 (Matriptase-2), MT-SP1 / matrix proteinase, TMPRSS2, TMPRSS3 and / or TMPRSS4); etc. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the group consisting of uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Casparase-1, Casparase-2, Casparase-3, Casparase-4, Casparase-5, Casparase-6, Casparase-7, Casparase-8, Casparase-9, Casparase-10, Casparase-11, Casparase-12, Casparase-13, Casparase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the group consisting of uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, the protease cleavage site comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 86) and PLGLAG (SEQ ID NO: 87). ;

[0096] In some embodiments, LU further comprises a first linker (L1). In some embodiments, the first linker (L1) is at the C-terminus of the first cleavage site (CS1) (e.g., the first protease cleavage site). In some embodiments, LU comprises a (CS1)-L1 structure from the N-terminus to the C-terminus. In some embodiments, LU further comprises a second linker (L2). In some embodiments, L2 is at the C-terminus of the second cleavage site. In some embodiments, LU comprises a (CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus. In some embodiments, L1 and L2 are any suitable linkers known in the art (e.g., flexible linkers), including but not limited to, for example, glycine polymers (G)n, wherein n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymers (GS)n, wherein n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as GGGGS (SEQ ID NO: 108), GGGGT (SEQ ID NO: 78), SGGS (SEQ ID NO: 79), GGSG (SEQ ID NO: 80), GGSGG (SEQ ID NO: 81), GSGSG (SEQ ID NO: 82), GSGGG (SEQ ID NO: 83), GGGSG (SEQ ID NO: 84), and / or GSSSG (SEQ ID NO: 85). NO:85); glycine-alanine polymer; alanine-serine polymer; and the like. The linker sequence can be of any length, such as about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymer or glycine-serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0097] In some embodiments, LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 10-16. SEQ ID NOs: 8 and 10-16 are provided in Table 3 below.

[0098] Table 3. Exemplary linker sequences (SEQ ID NOs: 8-36)

[0099] SEQ ID NO: Connection Unit (LU) sequence 8 SGRSAGGGGTPLGLAGSGGS 10 PLGLAGSGGS 11 GGGPLGLAGGGS 12 GGGPLGLAGGS 13 SGGGPLGLAGGS 14 SGRGPLGLAGGS 15 SGRGGPLGLAGGS 16 SGRGGGPLGLAGGS

[0100] In some embodiments, the shielding peptide (MP) comprises a (MU)-(LU) structure from N-terminus to C-terminus, wherein LU comprises a (CS1)-L1 or (CS1)-L1-(CS2)-L2 structure. In some embodiments, the shielding peptide of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-65.

[0101] In some embodiments, the shielding peptide (MP) comprises MU as set forth in any one of SEQ ID NOs: 1-7 and LU as set forth in any one of SEQ ID NOs: 8-16. In some embodiments, the MP comprises a sequence as set forth in any one of SEQ ID NOs: 17-35. SEQ ID NOs: 17-35 are provided in Table 4 below.

[0102] Table 4. Exemplary shielding peptide sequences (SEQ ID NOs: 17-35)

[0103]

[0104] * The N-terminal unit sequence is in plain text; The masking sequence is bold underline; Connected cells are in bold

[0105] In some embodiments, the masked antibody (which is also referred to herein as a "masked anti-CD137 antibody") comprises the VH shown in SEQ ID NO: 52 and the VL shown in SEQ ID NO: 53. In some embodiments, the masked antibody (which is also referred to herein as a "masked anti-CD137 antibody") comprises the VH shown in SEQ ID NO: 54 and the VL shown in SEQ ID NO: 55. In some embodiments, the masked antibody (which is also referred to herein as a "masked anti-CD137 antibody") comprises the VH shown in SEQ ID NO: 56 and the VL shown in SEQ ID NO: 57. In some embodiments, the masked anti-CD137 antibody comprises the VH shown in SEQ ID NO: 52 and the VL shown in any one of SEQ ID NOs: 58-76. SEQ ID NOs: 52-76 are provided in Tables 5A and 5B below.

[0106] Table 5A. VH and VL sequences of exemplary anti-CD137 antibodies

[0107]

[0108] Table 5B. VH and VL sequences of exemplary masked anti-CD137 antibodies* (N to C terminus)

[0109]

[0110]

[0111] * The N-terminal unit is plain text, The masking unit is bold bottom line , junction units are in bold, VL sequences are in italic text

[0112] In some embodiments, the masked anti-CD137 antibody comprises an MP comprising SEQ ID NO: 34; a VH comprising a CDR-H1 set forth in TGGVGVG (SEQ ID NO: 36), a CDR-H2 set forth in LIDWADDKYYSPSLKS (SEQ ID NO: 37), and a CDR-H3 set forth in GGSDTVIGDWFAY (SEQ ID NO: 38); and a VL comprising a CDR-L1 set forth in RASQSIGSYLA (SEQ ID NO: 39), a CDR-L2 set forth in DASNLET (SEQ ID NO: 40), and a CDR-L3 set forth in QQGYYLWT (SEQ ID NO: 41). In some embodiments, the masked antibody comprises an MP comprising SEQ ID NO: 34; a VH comprising SEQ ID NO: 52, and a VL comprising SEQ ID NO: 53. In some embodiments, the masked antibody comprises a VH comprising SEQ ID NO: 52 and a VL comprising SEQ ID NO: 58.

[0113] In some embodiments, the shielded anti-CD137 antibody comprises a full-length antibody light chain, for example, a kappa or lambda light chain. Additionally or alternatively, in some embodiments, the anti-CD137 antibody comprises a full-length antibody heavy chain. The heavy chain of the antibody may be any class, such as IgG, IgM, IgE, IgA or IgD. In some embodiments, the antibody heavy chain is an IgG class, such as an IgG1, IgG2, IgG3 or IgG4 subclass. Methods known in the art can be used to convert the antibody heavy chain from one class or subclass to another class or subclass. In some embodiments, the shielded anti-CD137 antibody is or comprises a full-length antibody containing an Fc region (for example, a human Fc region or a variant thereof). In some embodiments, the human Fc region is a human IgG1 Fc region, a human IgG2 Fc region, a human IgG4 Fc region, or a variant of any of the foregoing. In some embodiments, relative to the wild-type human Fc region from which the Fc region variant is derived, the Fc region variant comprises one or more amino acid substitutions, insertions or deletions. In some embodiments, the masked anti-CD137 antibody comprises a human IgG1 Fc region variant. In some embodiments, the IgG1 Fc variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant to FcγRIIb. In some embodiments, the human IgG1 Fc region variant comprises a substitution selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F, wherein the amino acid numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85). The aforementioned substitutions are described in Chu et al. (2008) Mol Immunol. 45 (15): 3926-33. Additionally or alternatively, in some embodiments, human IgG1 Fc region variants comprise a member selected from the group consisting of E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G237D, H268D, P238D and P271G; G23 7D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and substitutions of the group consisting of E233D, G237D, P238D, H268D, P271G and A330R, wherein the amino acid numbering is according to the EU index. The aforementioned substitutions are described in Mimoto et al. (2013) Protein Eng Des Sel. 26(10): 589-98.Additionally or alternatively, in some embodiments, human IgG1 Fc region variants comprise S2657A substitutions (see Buschor et al. (2014) Int Arch Allergy Immunol. 163(3): 206-14), wherein amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, human IgG1 Fc region variants comprise T437R and / or K248E substitutions (see Zhang et al. (2017) MAbs. 9(7): 1129-1142), wherein amino acid numbering is according to the EU index. In some embodiments, the shielded anti-CD137 antibody comprises human IgG4 Fc region variants. In some embodiments, IgG4 Fc region variants comprise one or more amino acid substitutions that increase the affinity of the Fc variant to FcγRIIb. In some embodiments, the human IgG4 Fc region variant comprises a substitution selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the human IgG4 Fc region variant comprises a substitution selected from the group consisting of E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G237D, H268D, P238D and P271G; G23 7D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and substitutions from the group consisting of E233D, G237D, P238D, H268D, P271G and A330R, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the human IgG4 Fc region variant comprises an S2657A substitution, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the human IgG1 Fc region variant comprises a T437R and / or a K248E substitution, wherein the amino acid numbering is according to the EU index.

[0114] In some embodiments, the masked anti-CD137 antibody comprises a masking peptide of SEQ ID NO: 34, a VH domain shown in SEQ ID NO: 52, and a VL domain shown in SEQ ID NO: 53. In some embodiments, the masked anti-CD137 antibody further comprises a human IgG1 domain or a variant thereof containing one or more substitution mutations. In some embodiments, the IgG1 variant comprises an IgG1 variant selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G2 37D, H268D, P238D and P271G;G237D, P238D, P271G and A330R;E233D, H268D, P238D, P271G and A330R;G237D, H268D, P238D, P271G and A330R;And E233D, G237D, P238D, H268D, P271G and A330R;S2657A;T437R;K248E;And the substitution of the group consisting of T437R and K248E, wherein the amino acid numbering is according to the EU index.In some embodiments, the shielded anti-CD137 antibody further comprises a human IgG4 domain or a variant thereof containing one or more substitution mutations. In some embodiments, the IgG4 variant comprises an IgG4 variant selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G2 37D, H268D, P238D and P271G; G237D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and E233D, G237D, P238D, H268D, P271G and A330R; S2657A; T437R; K248E; and T437R and K248E, wherein the amino acid numbering is according to the EU index.

[0115] In some embodiments, the masked anti-CD137 antibody comprises a heavy chain constant region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 111-118. See Table 6 below.

[0116] Table 6. Exemplary heavy chain constant region sequences

[0117]

[0118]

[0119]

[0120] In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising any one of SEQ ID NOs: 88-95. Additionally or alternatively, in some embodiments, the masked anti-CD137 antibody comprises a light chain comprising any one of SEQ ID NOs: 96-109 and 119-122.

[0121] In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 109. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 92 or 93 and a light chain comprising SEQ ID NO: 109. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 109. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO: 109. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO: 96. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 92 or 93 and a light chain comprising SEQ ID NO: 96. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 97. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 98. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 119. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 100. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 120. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 121. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 102. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 103. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 98. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 1.In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 99. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 100. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 99. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO: 99. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 101. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO: 101. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 102. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 90 or 91 and a light chain comprising SEQ ID NO: 101. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO: 104. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 105. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 106. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 88 or 89 and a light chain comprising SEQ ID NO: 107. In some embodiments, the masked anti-CD137 antibody comprises a heavy chain comprising SEQ ID NO: 94 or 94 and a light chain comprising SEQ ID NO: 122. The amino acid sequences of SEQ ID NOs: 88-95-109 and 119-122 are provided in Tables 7A and 7B below.

[0122] Table 7A. Heavy and light chain sequences of exemplary non-masked anti-CD137 antibodies (N to C terminus)

[0123]

[0124]

[0125]

[0126] For LC, VL sequences are in italic text, CL is double bottom line text

[0127] For HC, VH sequences are in italic text, CH is double bottom line text

[0128] Table 7B. Heavy and light chain sequences of exemplary masked anti-CD137 antibodies (N to C terminus)

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] · For LC, the N-terminal unit is plain text, The masking unit is bold bottom line, Linking units are in bold, VL sequences are in italic text,

[0148] For HC, VH sequences are in italic text,

[0149] In some embodiments, the term "shielded anti-CD137 antibody" refers to an antibody fragment, e.g., a shielded antigen-binding fragment of a shielded anti-CD137 antibody. In some embodiments, the antibody fragment is or comprises Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv (see Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), (scFv)2, linear antibody, single-chain antibody, single-domain antibody (nanobody, nanobody) VHH, mini antibody (minibody) or bispecific antibody (diabody).

[0150] In some embodiments, the shielded anti-CD137 antibodies described herein cross-react with CD137 from different species, thereby allowing shielded anti-CD137 antibodies to be used in preclinical and clinical studies. In some embodiments, shielded anti-CD137 antibodies described herein are activated (e.g., after the shielded antibody is activated by cleavage (e.g., protease cleavage)) in conjunction with two or more of human CD137, cynomolgus monkey CD137, murine (mouse) CD137 and / or rat CD137. In some embodiments, shielded anti-CD137 antibodies are activated (e.g., after the shielded antibody is activated by cleavage (e.g., protease cleavage)) in conjunction with human CD137, cynomolgus monkey CD137, murine (mouse) CD137 and rat CD137.

[0151] In some embodiments, the masked anti-CD137 antibodies described herein are context-dependent (e.g., activated (can only bind to its target) in certain contexts (such as in a protease-rich tumor microenvironment)). In some embodiments, the masked anti-CD137 antibodies described herein provide improved safety over more traditional non-shielded antibodies (e.g., showing reduced toxicity, not inducing significant changes in the weight of many organs, not changing liver histopathology, hematology and / or blood biochemistry, etc.). In some embodiments, the masked anti-CD137 antibodies described herein exhibit similar pharmacokinetic properties to traditional non-shielded anti-CD137 antibodies (e.g., having similar in vivo half-lives). In some embodiments, the masked anti-CD137 antibodies described herein exhibit improved pharmacokinetic properties (e.g., having longer in vivo half-lives) compared to more traditional non-shielded anti-CD137 antibodies.

[0152] In some embodiments, the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) of the masked anti-CD137 antibodies described herein form an antigen binding domain (ABD) that binds to hCD137. In some embodiments, the shielding unit (MU) of the masked anti-CD137 antibodies described herein is bound to the ABD and binds to hCD137 compared to the corresponding anti-CD137 antibody lacking the MU and / or reduces or inhibits the binding of the masked anti-CD137 antibody to hCD137 compared to the binding of the ABD to hCD137. In some embodiments, the shielding unit has a shielding efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, etc., including any ranges between these values) prior to removal of the MU from the shielded anti-CD137 antibody. In some embodiments, shielding efficiency is measured as the difference in affinity of a shielded anti-CD137 antibody comprising a shielding unit (MU) to hCD137 (e.g., before activation of the shielded antibody) relative to an anti-CD137 antibody lacking the MU to hCD137. In some embodiments, shielding efficiency is measured as the difference in affinity of a shielded anti-CD137 antibody comprising a MU to hCD137 (e.g., before activation of the shielded antibody by cleavage (e.g., protease cleavage)) relative to an unshielded anti-CD137 antibody (e.g., after activation of the shielded antibody by cleavage (e.g., protease cleavage)). In some embodiments, shielding efficiency can be measured by the EC of a shielded anti-CD137 antibody comprising a MU (e.g., before activation). 50 divided by the EC of the corresponding anti-CD137 antibody lacking the shielding peptide or the shielding unit 50 In some embodiments, EC 50 Measured by ELISA. In some embodiments, the shielding unit (MU) of the masked anti-CD137 antibody binds to the ABD and prevents the binding of the masked anti-CD137 polypeptide to hCD137.

[0153] In some embodiments, the affinity of the masked anti-CD37 antibodies of the present disclosure is increased by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more, including any ranges between the foregoing values) when the shielding unit is removed from the antibody (e.g., after activation by treatment with one or more proteases that cleave within the linking unit), compared to the corresponding anti-CD137 antibody without the shielding peptide or shielding unit. In some embodiments, the EC of the masked anti-CD137 antibodies described herein is 50 At least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more, including any ranges between the foregoing values) after activation by treatment with one or more proteases that cleave within the linking unit (e.g., as measured by ELISA or FACS assays).

[0154] In some embodiments, when the shielding unit is bound to the ABD of a shielded anti-CD137 antibody described herein, the K of the antibody to its target is D is about the K of the antibody when the shielding unit is removed from the ABD of the shielded anti-CD137 antibody (e.g., after treatment with a protease that cleaves within the linking unit). D In some embodiments, when the shielding unit is bound to the ABD of a shielded anti-CD137 antibody described herein, the K of the antibody to its target is about 2 times (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any range between the foregoing values). D The K of the corresponding anti-CD137 antibody lacking the shielding peptide or shielding unit is about Dtwice (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any ranges between the foregoing values).

[0155] In some embodiments, the shielding unit sterically hinders the shielded binding polypeptide from binding to its target and / or allosterically hinders the shielded binding polypeptide from binding to its target.

[0156] In some embodiments, the dissociation constant of the shielding unit for a shielded anti-CD137 antibody described herein for ABD is higher than the dissociation constant of the shielded anti-CD137 antibody for hCD137 (when the shielded anti-CD137 antibody is in an activated form, e.g., after protease treatment). In some embodiments, the dissociation constant of the shielding unit for the ABD of the shielded antibody described herein is about 2 (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any ranges between the foregoing values) times higher than the dissociation constant of the shielded anti-CD137 antibody for hCD137 (when the shielded anti-CD37 antibody is in an activated form, such as after treatment with a protease). In some embodiments, the dissociation constant of the shielding unit to the shielded anti-CD137 antibody described herein to the ABD is approximately equal to the dissociation constant of the shielded anti-CD137 antibody to hCD137 (when the shielded anti-CD137 antibody is in an activated form, such as after treatment with a protease). In some embodiments, when the shielded anti-CD137 antibody has not yet been activated (e.g., after treatment with one or more proteases that are cleaved within the connecting unit), the shielding unit (MU) is bound to the ABD of the shielded anti-CD137 antibody described herein and prevents the antibody from binding to hCD137. In some embodiments, the cleavage of the polypeptide within the activation-induced cleavage site. In some embodiments, the conformational change of the activation-induced polypeptide (e.g., substitution of the shielding unit (MU)) causes the shielding peptide to no longer prevent the polypeptide from binding to its target.

[0157] The shielded antibodies described herein may be further modified. In some embodiments, those shielded antibodies are linked to additional molecular entities. Examples of additional molecular entities include pharmaceutical preparations, peptides or proteins, detection agents or labels, and antibodies.

[0158] In some embodiments, the activatable binding polypeptide of the present disclosure is linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents and radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, miramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., dichloromethyl diethylamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, milramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 and Lutetium 177 Methods for linking polypeptides to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfide, and peptide-containing linkers. For further discussion of linkers and methods for attaching therapeutic agents to antibodies, see, e.g., Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0159] Nucleic acids, vectors, host cells and recombinant production methods for producing masked anti-CD137 antibodies

[0160] Another aspect of the present disclosure provides one or more isolated nucleic acid molecules, which include nucleotide sequences encoding the amino acid sequences of shielded anti-CD137 antibodies described herein. The amino acid sequence encoded by the nucleotide sequence can be any part of the shielded anti-CD137 antibodies (such as CDR, a sequence comprising one, two or three CDRs, a heavy chain variable region, a light chain variable region), or can be a full-length heavy chain or a full-length light chain. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0161] In some embodiments, the invention provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence, e.g., a heavy chain variable region and / or a light chain variable region of a masked anti-CD137 antibody described herein, or, e.g., a full-length heavy chain or full-length light chain of a masked anti-CD137 antibody described herein.

[0162] Any suitable molecular biology technique (eg, PCR amplification or cDNA cloning technique) can be used to obtain the nucleic acid of the present invention. For the masked anti-CD137 antibody obtained from the library screening, the nucleic acid encoding the antibody can be recovered from the library.

[0163] By encoding V H The DNA encoding the heavy chain constant region (CH1, CH2 and CH3) is operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). H The isolated DNA of the heavy chain region is converted into a full-length heavy chain gene. The sequence of human heavy chain constant region genes is known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH publication No. 91-3242), and DNA fragments covering 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 IgG4 or IgG2 constant region without ADCC effect. The IgG4 constant region sequence can be any of the various alleles or allotypes known to exist in different individuals. These allotypes represent naturally occurring amino acid substitutions in the IgG4 constant region. For the Fab fragment heavy chain gene, the encoding V H The DNA is operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0164] By encoding V L The DNA encoding the light chain constant region CL is operably linked to another DNA molecule encoding the V LThe isolated DNA of the region is converted into a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and the DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. In some embodiments, the shielded anti-CD137 antibody comprises the light chain constant region shown in SEQ ID NO: 111, which is provided as follows: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 111)

[0165] To generate scFv genes, the V H and V L The DNA fragment encoding the flexible linker is operably linked to another fragment encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 128), such that V H and V L The sequence can be expressed as a V L and V H The invention relates to a continuous single-chain protein containing a cleavage region (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990)).

[0166] The present disclosure further provides a vector comprising one or more nucleic acid molecules described herein. In some embodiments, the vector is an expression vector that can be used to express a shielded anti-CD137 antibody or a shielded antigen-binding fragment of such an antibody. In some embodiments, a vector is provided herein, wherein the first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and the second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises a polynucleotide encoding a heavy chain variable region as described herein and a light chain variable region as described herein.

[0167] To express the binding molecules of the present disclosure, the DNA encoding part or full-length light chain and heavy chain is inserted into an expression vector so that the DNA molecule is operably connected to the transcription and translation control sequence. In this case, the term "operably connected" means that the antibody gene is connected to the vector so that the transcription and translation control sequences in the vector play their predetermined functions of regulating the transcription and translation of the DNA molecule. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. Antibody light chain genes and antibody heavy chain genes can be inserted into a separate vector, or more generally, two genes can be inserted into the same expression vector. Antibody genes are inserted into expression vectors by any suitable method (such as connection of complementary restriction sites on antibody gene fragments and vectors, or connection of DNA based on homology recombination). The light chain variable region and heavy chain variable region of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype and subclass by inserting them into expression vectors encoding the heavy chain constant region and light chain constant region of the desired isotype and subclass, so that the VH segment is operably connected to one or more CH segments in the vector, and the VL segment is operably connected to the CL segment in the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide that helps the antibody chain to be secreted from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in the same frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0168] In addition to the antibody chain gene, the expression vector of the present disclosure generally also carries a regulatory sequence for controlling the expression of the antibody chain gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. The regulatory sequence is described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the desired protein expression level, etc. Examples of regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyomavirus. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters may be used. In addition, regulatory elements are composed of sequences from different sources, such as the SR promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0169] In addition to antibody chain genes and regulatory sequences, expression vectors may also carry additional sequences, such as enhancer components, transcription termination sequences, sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. Selectable marker genes may help select host cells into which vectors have been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all completed by Axel et al.). For example, typically, selectable marker genes confer resistance to drugs such as G418, hygromycin or methotrexate to host cells into which vectors have been introduced. Selectable marker genes include dihydrofolate reductase (DHFR) genes (used in dhfr-host cells in conjunction with methotrexate selection / amplification) and neo genes (selected for G418).

[0170] For the expression of light and heavy chains, one or more expression vectors encoding heavy and light chains are transfected into host cells by any suitable technique. The various forms of the term "transfection" are intended to cover a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express the shielded anti-CD137 antibodies described herein in prokaryotic or eukaryotic host cells, it is most typical to express antibodies in eukaryotic cells, such as mammalian host cells.

[0171] The present disclosure also provides a host cell containing a nucleic acid molecule or a vector provided by the present disclosure. The host cell can be virtually any cell for which an expression vector is available. It can be, for example, a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, and can be a prokaryotic cell such as a bacterial cell. The introduction of a recombinant nucleic acid construct into a host cell can be achieved by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

[0172] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0173] Mammalian host cells for expressing the binding molecules of the present disclosure include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol. 159:601-621 (1982)), NS0 myeloma cells, COS cells, and Sp2 cells. In particular, for use with NS0 myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.

[0174] The shielded anti-CD137 antibodies (or antigen-binding fragments thereof) disclosed herein can be produced using any known method in the art. Exemplary techniques for antibody production are shown in U.S. Pat. No. 4,816,567; however, this exemplary technique is provided for illustrative purposes only and is not intended to be limiting. When a nucleic acid or expression vector encoding a shielded anti-CD137 antibody is introduced into a host cell, the shielded anti-CD137 antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell grows. Therefore, in some embodiments, a method for producing a shielded anti-CD137 antibody as described herein is provided, the method comprising culturing a host cell comprising one or more nucleic acids or vectors encoding a shielded anti-CD137 antibody (e.g., provided above) under suitable conditions for expressing the shielded antibody. In some embodiments, the method further comprises recovering the shielded anti-CD137 antibody from the host cell (or host cell culture medium). Any suitable protein purification method can be used to recover the shielded anti-CD137 antibody from the culture medium.

[0175] Pharmaceutical composition

[0176] In other aspects, the present disclosure provides a composition comprising one or more shielded anti-CD137 antibodies described herein. In some embodiments, the composition is a pharmaceutical composition comprising a shielded anti-CD137 antibody described herein and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0177] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a polypeptide (e.g., a shielded antibody). The carrier may be an anti-adhesive, a binding agent, a coating agent, a disintegrant, a filler or a diluent, a preservative (such as an antioxidant, an antibacterial agent or an antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline and isotonic agents, such as sugars, polyols, sorbitol and sodium chloride.

[0178] The composition can be in any suitable form, such as liquid, semi-solid and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable solutions and insoluble solutions), microemulsions, liposomes, dispersions or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules and powders. A specific form of the composition suitable for delivering shielded anti-CD137 antibodies is a sterile liquid for injection or infusion, such as a solution, suspension or dispersion. A sterile solution can be prepared by incorporating the shielded anti-CD137 antibody into a suitable carrier in the required amount, followed by sterile microfiltration. Dispersions can be prepared by incorporating the shielded anti-CD137 antibody into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of a sterile powder for preparing a sterile liquid, the preparation method includes vacuum drying and freeze drying (lyophilization) to obtain a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution. The various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0179] The relative amount of the masked anti-CD137 antibody included in the composition will vary depending on many factors, such as the specific polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of the masked anti-CD137 antibody in a single dosage form will generally be an amount that produces a therapeutic effect, but may also be a lesser amount. Typically, this amount will be in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% relative to the total weight of the dosage form.

[0180] In addition to shielded anti-CD137 antibodies, one or more additional therapeutic agents may also be included in the composition. Examples of additional therapeutic agents are described in WO 2019 / 037711, which is incorporated herein by reference in its entirety. The suitable amount of the additional therapeutic agent included in the composition can be easily selected by those skilled in the art, and will vary depending on many factors, such as the specific agent and carrier used, dosage form, and desired release and pharmacodynamic characteristics. The amount of the additional therapeutic agent included in a single dosage form will generally be that amount of the agent that produces a therapeutic effect, but may also be a smaller amount.

[0181] Any of the masked anti-CD137 antibodies and / or compositions (eg, pharmaceutical compositions) described herein can be used to prepare a medicament (eg, a medicament for treating or delaying progression of cancer in a subject in need thereof).

[0182] Treatment

[0183] The shielded anti-CD137 antibodies and pharmaceutical compositions described herein can be used for therapeutic purposes, such as treating cancer or enhancing the efficacy of other cancer therapies. Therefore, in other aspects, the present disclosure provides methods of using shielded anti-CD137 antibodies or pharmaceutical compositions. In one aspect, the present disclosure provides a method of treating a subject (e.g., a human subject) cancer, comprising administering to the subject an effective amount of a shielded anti-CD137 antibody. In some embodiments, the cancer is a solid tumor cancer (e.g., breast cancer, liver cancer, colorectal cancer, colon cancer, etc.).

[0184] In the practice of treatment methods, the shielded anti-CD137 antibodies described herein can be administered alone, for example, as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Therefore, on the other hand, the present disclosure provides a combination therapy, which includes a binding molecule combined with one or more additional therapies or therapeutic agents for separate, sequential or simultaneous administration. In some embodiments, the term "additional therapy" refers to a therapy that does not use shielded anti-CD137 antibodies as therapeutic agents. In some embodiments, the term "additional therapeutic agent" refers to any therapeutic agent other than the shielded anti-CD137 antibodies described herein. In some embodiments, the present invention provides a method for treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of shielded anti-CD137 antibodies described herein and an effective amount of anti-PD-1 antibodies (e.g., anti-human PD-1 antibodies). In some embodiments, the present invention provides a method for treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of shielded anti-CD137 antibodies described herein and an effective amount of anti-CTLA4 antibodies (e.g., anti-human CTLA4 antibodies). In some embodiments, the anti-CTLA4 antibody is a shielded anti-CTLA4 antibody. In some embodiments, the present invention provides a method for treating a subject (e.g., a human subject) cancer, comprising administering to the subject an effective amount of a shielded anti-CD137 antibody as described herein and an effective amount of a bispecific antibody binding to HER2 (e.g., human HER2) and CD3 (e.g., human CD3). In some embodiments, the bispecific antibody binding HER2 and CD3 is a shielded bispecific antibody binding HER2 and CD3. In some embodiments, the present invention provides a method for treating a subject (e.g., a human subject) cancer, comprising administering to the subject an effective amount of a shielded anti-CD137 antibody as described herein and an effective amount of a bispecific T cell adapter (TCE) targeting CD3 (e.g., human CD3) and an antigen expressed on the surface of a solid tumor cancer cell. In some embodiments, the antigen expressed on the surface of a solid tumor is HER2 (e.g., human HER2). In some embodiments, the TCE targeting CD3 and HER2 comprises three polypeptide chains, wherein the first polypeptide chain comprises an antibody heavy chain, the second polypeptide chain comprises an antibody light chain, and the third polypeptide chain comprises scFv-Fc, wherein the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, wherein the first and second polypeptide chains associate to form a HER2 binding arm, wherein the third polypeptide chain binds CD3, and wherein the Fc domain of the anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form the TCE. In some embodiments, the TCE is a shielded TCE (e.g., wherein the portion of the TCE binding to CD3 and / or the portion of the TCE binding to HER2 is shielded).In some embodiments, the first polypeptide chain comprises SEQ ID NO: 125, the second polypeptide chain comprises SEQ ID NO: 124, and the third polypeptide chain comprises SEQ ID NO: 126. In some embodiments, the cancer is a solid tumor (e.g., colon cancer, breast cancer, liver cancer, colorectal cancer, or colon cancer).

[0185] In some embodiments of any of the treatment methods described herein, the masked anti-CD137 antibody comprises a masking peptide of SEQ ID NO: 34, a VH domain shown in SEQ ID NO: 52, and a VL domain shown in SEQ ID NO: 53. In some embodiments, the masked anti-CD137 antibody further comprises a human IgG1 domain or a variant thereof containing one or more substitution mutations. In some embodiments, the IgG1 variant comprises an IgG1 variant selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G2 37D, H268D, P238D and P271G;G237D, P238D, P271G and A330R;E233D, H268D, P238D, P271G and A330R;G237D, H268D, P238D, P271G and A330R;And E233D, G237D, P238D, H268D, P271G and A330R;S2657A;T437R;K248E;And the substitution of the group consisting of T437R and K248E, wherein the amino acid numbering is according to the EU index.In some embodiments, the shielded anti-CD137 antibody further comprises a human IgG4 domain or a variant thereof containing one or more substitution mutations. In some embodiments, the IgG4 variant comprises an IgG4 variant selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G2 37D, H268D, P238D and P271G; G237D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and E233D, G237D, P238D, H268D, P271G and A330R; S2657A; T437R; K248E; and T437R and K248E, wherein the amino acid numbering is according to the EU index.

[0186] Kits and Products

[0187] In another aspect, a kit is provided, comprising one or more shielded anti-CD137 antibodies described herein. In some embodiments, the kit further comprises a package insert comprising instructions for using the shielded anti-CD137 antibodies. In some embodiments, the article or kit comprises a container comprising one or more shielded anti-CD137 antibodies or compositions described herein. In certain embodiments, the article or kit comprises a container comprising nucleic acids encoding one (or more) shielded anti-CD137 antibodies described herein. In some embodiments, the kit comprises cells of a cell line producing a shielded anti-CD137 antibody described herein. In some embodiments, the kit comprises one or more CD137 (e.g., human CD137, cynomolgus monkey CD137, mouse CD137, rat CD137, or any of the foregoing fragments) or CD137 + Positive Controls for Cells. In some embodiments, the kit includes a negative control, such as a surface or solution that contains little or no CD137, or cells that do not express CD137.

[0188] In certain embodiments, the article or kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the label or package insert indicates that the shielded anti-CD137 antibody (e.g., according to the methods provided herein) is used to treat solid tumors (e.g., breast cancer, liver cancer, colorectal cancer, colon cancer, etc.) in subjects in need (e.g., human subjects). Suitable containers include, for example, bottles, vials, syringes, IV solution bags, test tubes, etc. The container can be formed of a variety of materials, such as glass or plastic. The container holds the shielded anti-CD137 antibody used herein (or a composition comprising this shielded antibody), which is administered by itself or in combination with another composition, and can effectively treat, delay the process, and / or prevent cancer in subjects (e.g., human subjects). The container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used to treat breast cancer, liver cancer, colorectal cancer, or colon cancer in subjects (e.g., human subjects).

[0189] In addition, the article or kit may include (a) a first container containing a composition, wherein the composition includes a shielded anti-CD137 antibody (or an immunologically active fragment thereof) described herein; and (b) a second container containing a composition, wherein the composition includes an additional cytotoxic agent or an additional therapeutic agent. In some embodiments, the second container contains a composition comprising an anti-PD-1 antibody (e.g., an anti-human PD-1 antibody), and an article containing a label or package insert indicating that the shielded anti-CD137 antibody and anti-PD-L1 are used to treat solid tumors (e.g., breast cancer, liver cancer, colorectal cancer, colon cancer, etc.) in a subject in need thereof (e.g., a human subject), for example, according to the methods provided herein. In some embodiments, the second container contains a composition comprising an anti-CTLA4 antibody (e.g., an anti-human CTLA4 antibody, such as a shielded anti-CTLA-4 antibody), and an article of manufacture comprising a label or package insert indicating that the shielded anti-CD137 antibody and the anti-CTLA4 antibody (e.g., a shielded anti-CTLA-4 antibody) are used to treat a solid tumor (e.g., breast cancer, liver cancer, colorectal cancer, colon cancer, etc.) in a subject (e.g., a human subject) in need thereof, e.g., according to the methods provided herein. In some embodiments, the second container contains a composition comprising a bispecific antibody that binds HER2 (e.g., human HER2) and CD3 (e.g., human CD3) (e.g., a shielded bispecific antibody that binds HER2 and CD3), and an article of manufacture comprising a label or package insert indicating that the shielded anti-CD137 antibody and the bispecific antibody that binds HER2 and CD3 (e.g., a shielded bispecific antibody that binds HER2 and CD3) are used to treat a subject (e.g., a human subject) with a solid tumor (e.g., breast cancer, liver cancer, colorectal cancer, colon cancer, etc.), for example, according to the methods provided herein. In some embodiments, the second container contains a composition comprising a bispecific T cell engager (TCE) that binds CD3 (e.g., human CD3) and an antigen expressed on the surface of a solid tumor cancer cell (e.g., a shielded bispecific T cell engager that binds CD3 and an antigen expressed on the surface of a solid tumor cancer cell). In some embodiments, the antigen expressed on the surface of a solid tumor is HER2 (e.g., human HER2). In some embodiments, the TCE targeting CD3 and HER2 comprises three polypeptide chains, wherein the first polypeptide chain comprises an antibody heavy chain, the second polypeptide chain comprises an antibody light chain, and the third polypeptide chain comprises scFv-Fc, wherein the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, wherein the first and second polypeptide chains associate to form a HER2 binding arm, wherein the third polypeptide chain binds CD3, and wherein the Fc domain of the anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form the TCE. In some embodiments, the TCE is a shielded TCE (e.g., wherein the portion of the TCE binding to CD3 and / or the portion of the TCE binding to HER2 is shielded).In some embodiments, the first polypeptide chain comprises SEQ ID NO: 125, the second polypeptide chain comprises SEQ ID NO: 124, and the third polypeptide chain comprises SEQ ID NO: 126. In some embodiments, the article of manufacture comprises a label or package insert indicating that a masked anti-CD137 antibody and a TCE that binds CD3 and an antigen expressed on the surface of a solid tumor cancer cell (e.g., a masked bispecific T cell engager that binds CD3 and an antigen expressed on the surface of a solid tumor cancer cell (e.g., HER2)) is used to treat a solid tumor (e.g., breast cancer, liver cancer, colorectal cancer, colon cancer, etc.) in a subject (e.g., a human subject) in need thereof, e.g., according to the methods provided herein.

[0190] In addition, the article of manufacture may further comprise other containers comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0191] The previous written description is considered sufficient to enable those skilled in the art to implement the present disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. In fact, in addition to those modifications shown and described herein, various modifications of the present disclosure will become apparent to those skilled in the art based on the previous description and fall within the scope of the appended claims.

[0192] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0193] Example

[0194] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors consider their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0195] Example 1: Construction and validation of shielded antibodies targeting CD137

[0196] FACS-based screening of CD137 antibody-shielding peptides

[0197] A total of 1×10 8 Yeast cells were screened for shielding peptides against the parent anti-CD137 antibody TY21242, which comprises the heavy chain variable region (VH) shown in SEQ ID NO:52 and the light chain variable region (VL) sequence shown in SEQ ID NO:53, which is provided below. (CDR sequences are in bold).

[0198]

[0199] In this example, the shielding units from CPL were directly fused to the N-terminus of the light chain of the parent antigen-binding domain, and a yeast library displaying the fusion protein on the surface of yeast cells was constructed. To identify shielding peptide sequences that can effectively shield the parent anti-CD137 antibody, the yeast library was then subjected to several rounds of FACS-based screening: first, yeast clones with low binding to human CD137 were enriched, followed by protease treatment of the enriched yeast clones to remove the shielding units, and clones with high binding to the antigen were selected.

[0200] In short, for MOFLO Each round of sorting performed by the high-speed cell sorter, the yeast cells induced in the galactose medium were harvested, washed once with PBSA buffer (e.g., 1% BSA in PBS), and then incubated at room temperature for 1 hour with different concentrations of biotinylated CD137. The yeast cells were then washed twice with PBSA buffer and incubated at 4°C for 30 minutes with streptavidin (1:500 dilution) (eBioscience No. 2-4317-87) bound to erythrin (PE). After washing twice with PBSA buffer, the yeast cells were adjusted to 2-3OD / mL and subjected to sorting. 1nM biotinylated CD137-Fc was used in all 5 rounds of sorting, and weak binders were enriched in each round of sorting. Yeast cells showing weak binding from the 5th round were grown in glucose, and then induced in galactose medium, and treated with TEV protease (6U / OD cell) (inside) at 30°C for 2 hours. Yeast cells showing strong binders after protease treatment were purified. It is clear that TEV cleavage dramatically increases the population of cells that strongly bind to the antigen, indicating that the screening strategy is effective. Single clones from round 5 sorting were plated on selection medium and grown individually to further confirm cleavage-mediated activation antigen binding. In Fab format, the masked anti-CD137 antibody was selected to show little binding to the antigen (e.g., hCD137) in the presence of a masking peptide. However, when the yeast cells were treated with TEV protease to remove the masking peptide, binding to the antigen increased dramatically.

[0201] After 5 rounds of sorting, plasmids were extracted from these clones, and the shielding unit sequence was confirmed by DNA sequencing. To determine the amino acid sequence of the shielding peptide, shuttle plasmids were extracted from selected yeast clones (Generay numbering GK2002-200), and transformed into competent Escherichia coli cells. Plasmids were prepared, and the region encoding the shielding peptide was sequenced and compared. The sequence of the shielding peptide can be divided into several groups, which indicates that significant enrichment is produced by multiple rounds of sorting. The shielding unit sequences and connecting unit sequences of several antibodies are listed in Table A1. The complete sequence of the shielding peptide is shown in Table A2. The shielding peptide includes an N-terminal unit (i.e., EVGSY (SEQ ID NO: 77) amino acid sequence), a shielding unit, and a connecting unit from N-terminal to C-terminal. The antibody VH and VL sequences listed in Tables A1 and A2 are provided in Tables 5A and 5B.

[0202] Table A1: Sequences of masking units and connecting units

[0203]

[0204] Table A2: Sequences of masking peptides comprising an N-terminal unit, a masking unit and a linker unit*

[0206]

[0207] *N-terminal unit sequence is in plain text; The masking sequence is bold underline; Linker unit sequences are in bold.

[0208] IgG conversion and expression

[0209] The shielding unit and linking unit sequences of other shielded anti-CD137 antibodies derived from TY22586, TY22594, TY22595 and TY22599 are listed in Table B1. The complete sequence of the shielding peptide is shown in Table B2. The shielding peptide comprises an N-terminal unit (i.e., EVGSY (SEQ ID NO: 77) amino acid sequence), a shielding unit and a linking unit from N-terminus to C-terminus. The antibody VH and VL sequences listed in Tables B1 and B2 are provided in Tables 5A and 5B. The antibodies in Tables B1 and B2 are converted into IgG1 or IgG4 shielded antibodies, and each shielded antibody comprises one or two cleavage sites (e.g., uPA and / or matrix metalloproteinases (MMPs), such as MMP9). To enhance the binding of the masked antibodies in Tables B1 and B2 to FcγRIIb, S267E and L328F mutations (EU numbering) were introduced into the Fc region to generate TY25370, TY25371, TY25372, TY25366, TY25368 and TY25369. The heavy and light chains of each antibody were cloned separately into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific, catalog number K830001), and the masking peptide and cleavage peptide were fused to the N-terminus of the light chain, in the same manner as displayed on the surface of yeast in CPL.

[0210] Table B1: Sequences of shielding units and linking units derived from shielded anti-CD137 antibodies of TY22586, TY22594, TY22595 and TY22599

[0211]

[0212]

[0213] Table B2: Sequences of shielding peptides derived from shielded anti-CD137 antibodies of TY22586, TY22594, TY22595 and TY22599, comprising an N-terminal unit, a shielding unit and a linking unit

[0214]

[0215]

[0216] *N-terminal unit sequence is in plain text; The masking sequence is bold underline; Connected cells are in bold.

[0217] Each plasmid pair (that is, the N-terminus of each VL is fused to the peptide comprising the shielding sequence and the connecting unit) encoding the shielded anti-CD137 antibody VH and VL is transiently transfected into HEK293F cells. After six days, the supernatant is collected, clarified by centrifugation and filtration, and purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The shielded anti-CD137 antibody is eluted, neutralized, and the buffer is exchanged into 20mM histidine, pH 5.5 buffer. Protein concentration is determined by UV-spectrophotometry, and protein purity is analyzed by SDS-PAGE or SEC-HPLC under denaturation, reduction and non-reducing conditions. The expression level of the shielded anti-CD137 antibody in HEK293 cells is close to or lower than the parent antibody, and its purification yield after protein A resin is also close to the parent antibody, indicating that the presence of shielding peptides and cleavage peptides has no significant negative impact on the antibody expression in mammalian cells.

[0218] Measuring shading efficiency

[0219] The affinity of masked anti-CD137 antibodies to human CD137 displayed on the surface of yeast cells was also evaluated. Briefly, yeast cells were transfected with a plasmid expressing full-length human CD137 and then tagged with a C-terminal 3×Myc tag for identification of transfected cells. Transfected cells were transferred to a 1.5 mL tube, washed once with 1% PBSA buffer, centrifuged, and resuspended with 1 mL 1% PBSA buffer to a density of 0.2 OD 600 / mL, and aliquoted into the wells of a 96-well plate. The test antibody was pipetted into the 96-well plate containing the cells in a 3-fold serial dilution, incubated on ice for 1 hour (protected from light), washed once with 1% PBSA buffer, and incubated on ice for 30 minutes with 0.5μg / mL PE-conjugated mouse anti-human IgG FC and 2μg / mL mouse anti-myc-647. The cells were washed once and then analyzed by flow cytometry ( CytoFlex) analysis.

[0220] Multiple experiments were performed on some of the masked anti-CD137 antibodies, so these masked antibodies have two calculated masking efficiencies. The masking efficiency of each masked antibody was calculated by dividing the KD of the masked antibody binding to hCD137 by the KD of the parent antibody binding to hCD137. Figures 1A to 1DAs shown in Table C, each shielded antibody showed a dramatic reduction in binding rate to hCD137 compared to the parent antibody. The calculated shielding efficiency of the shielded antibodies ranged from 21 to 1131. The difference in shielding efficiency may be due to variations in measurement and data fitting, and the shielding efficiency of each shielded antibody may fall within the calculated range. These results indicate that multiple shielding peptides recognized by CPL, as part of a complete IgG molecule, retain their shielding efficiency when expressed in mammalian cells.

[0221] Table C: FACS-based measurement of KD of masked anti-CD137 antibodies before protease cleavage

[0222]

[0223]

[0224] Example 2: Activity of shielded anti-CD137 antibodies before and after removal of shielding peptides

[0225] The purified masked antibody is treated with a protease that recognizes the cleavage sequence in the linker unit. After treatment, the masked antibody is then tested to determine whether removal of the masking peptide can restore the activity of the antibody. As an example, 20 μg of TY25366 and TY25368 (0.5 mg / mL) are each treated with 1 μg of recombinant human MMP-9 (in-house) in a reaction buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 (w / v), pH 7.5). The reaction is carried out at 37°C for 24 hours. ELISA and FACS-based tests confirm that the masking peptide has been removed from the light chain. As Figures 2A to 2B and Table D, after removal of the masking peptide, the binding of the masked anti-CD137 antibodies to hCD137 was indistinguishable from that of the parental antibodies (eg, TY21242 and TY23310).

[0226] Table D: ELISA EC50 and EC50 of masked anti-CD137 antibodies after protease cleavage

[0227] FACS KD

[0228]

[0229] Example 3: Developability characteristics of masked anti-CD137 antibodies

[0230] For manufacturing purposes, it is essential that shielded anti-CD137 antibodies have good developability characteristics. As described in detail in this embodiment, different detections were performed with shielded antibodies expressed and purified in mammalian cells. The shielded antibody was adjusted to 1 mg / mL in 20 mM histidine (pH 5.5), and antibody quality was analyzed using an analytical size exclusion chromatography (SEC) using a Thermo U3000 with a Thermo DAD detector and XBridgeBEH SEC chromatographic column (7.8 mm × 300 mm) (Waters). For each assay, 40 μg of antibody was injected, and fractionation was performed at a flow rate of 0.7 mL / min with a buffer (50 mM phosphate in 300 mM sodium chloride, pH 6.8). As shown in Figures 3A to 3E, five accelerated stress tests were performed. Briefly, TY25366 and TY25368 were subjected to (1) 0, 3, and 6 freeze and thaw cycles; (2) storage at 40°C for 0, 7, 14, 21, and 28 days; (3) storage at room temperature in an acidic buffer (sodium acetate solution, pH 3.6) for 0 and 2 hours; (4) storage at 40°C in a 50 mM histidine, 300 mM NaCl, pH 7.0 buffer for 0 and 24 hours; and (5) storage in saline at room temperature for 6 hours and then at 4°C for 24 hours. The SEC characteristics of TY25366 and TY25368 after storage under accelerated storage conditions were similar to the SEC characteristics of TY25366 and TY25368 at T = 0 (i.e., before storage under the aforementioned conditions). The masked antibodies have not undergone extensive buffer optimization, and optimized buffers and excipients may further improve the stability of the masked antibodies. Together, these data demonstrate that the masked anti-CD137 antibody remains stable under a variety of stress conditions and therefore has a good developability profile.

[0231] Example 4: Binding of anti-CD137 antibodies TY24118 and TY24122 to FcγR

[0232] The binding of anti-CD137 antibodies TY24118 and TY24122 (i.e., the parent antibodies of masked antibodies TY25368 and TY25366, respectively) to Fcγ receptors was evaluated as follows. 2 mg / ml His-tagged recombinant human FcγR protein was captured by the anti-Penta-His sensor in Fortebio. Serial dilutions of the test antibody (i.e., TY24118 or TY24122) were then passed through the sensor for association, followed by a dissociation step in the electrophoresis buffer. The affinity of the antibodies for FcγR protein was calculated by steady-state analysis. As shown in Table E, TY24118 and TY24122 exhibited enhanced binding affinity to human FcγRIIa and FcγRIIb proteins compared to the corresponding wild-type antibodies (i.e., TY23310 and TY21242).

[0233] Table E: Binding affinity KD (nM) of TY24118 and TY24122 to human FcγR

[0234]

[0235] Example 5: Binding of masked anti-CD137 antibodies to target T cells

[0236] The binding of masked anti-CD137 antibodies to target T cells was assessed as follows. Human and cynomolgus monkey T cells were isolated from the peripheral blood of healthy donors. Mouse and rat T cells were isolated from the spleen. All T cells were cultured in the presence or absence of anti-CD3 / anti-CD28 stimulation. Activated T cells or unstimulated naive T cells were incubated with serially diluted test antibodies and corresponding anti-CD4 and anti-CD8 antibodies to gate T cell subsets. The binding of the test antibody was detected by FACS analysis with a fluorescently labeled anti-human IgG Fc secondary antibody. As shown in Figures 4A to D, only TY25368 cleaved by MMP9 showed positive staining on activated human T cells, cynomolgus monkey T cells, mouse T cells and rat T cells, consistent with the expression of CD137 induced in activated T cells. TY25368 cleaved by MMP9 did not show detectable binding to unstimulated naive T cells. TY25368 that was not cleaved did not show detectable binding to activated or naive T cells. These results indicate that masking of antibody TY25368 with a masking peptide prevents its binding to target T cells and that removal of the mask by protease cleavage restores the target cell binding activity of TY25368.

[0237] Example 6: Ligand blocking activity of masked anti-CD137 antibodies

[0238] The ligand blocking activity of the anti-CD137 masked antibody was evaluated as follows: ELISA plates were coated with 1 mg / ml recombinant human CD137 protein. Next, 2 mg / ml biotinylated recombinant human CD137 ligand was incubated with the CD137 pre-coated ELISA plates in the presence of serially diluted masked antibodies at 37°C for 1 hour. After washing, NeutrAvidin-HRP was added to the plates to detect the interaction between CD137 and its ligand. Figure 5 As shown, TY25368 cleaved by MMP9 showed strong inhibition of the interaction between CD137 and its ligand in a dose-dependent manner, with an IC50 of 4.46 nM. Uncleaved TY25368 showed no detectable inhibitory activity, similar to the isotype control antibody. These results indicate that TY25368 is able to block the interaction of the ligand with the CD137 receptor when cleaved.

[0239] Example 7: Activation of CD137-mediated cell signaling by masked anti-CD137 antibodies

[0240] Jurkat-CD137-NFκB-luciferase reporter cells were used to evaluate the stimulatory activity of anti-CD137 antibodies and their masked counterparts on CD137 receptor signaling. Briefly, reporter cells were cultured in the presence or absence of CHO-K1-hFcγRIIb cells as a cross-linking agent (E:CL=20:1, where "E" refers to Jurkat-CD137-NFκB-luciferase reporter cells and "CL" refers to CHO-K1-hFcγRIIb cells). Serial dilutions of test antibodies were added to the reporter cell system to evaluate their activity in stimulating downstream luciferase activity.

[0241] like Figures 6A to 6B As shown in Table F, in the presence of CHO-K1-hFcγRIIb cells as cross-linking agents, all anti-CD137 antibodies showed CD137 signaling activation. The shielded antibody TY25368 treated with MMP9 showed the strongest signal. The activity of the uncleaved shielded antibody TY25368 was much weaker than that of the cleaved form. Other clinical anti-CD137 antibodies were also compared in the test. Anti-CD137 antibodies AC1121 and AC1097 are described in WO 2019 / 036855 and US2020 / 00369776, which are incorporated herein by reference in their entirety. It is worth noting that in the absence of CHO-K1-hFcγRIIb cells as cross-linking agents, only AC1121 showed activity. All other anti-CD137 antibodies were inactive, indicating that their CD137 agonist activity was cross-linking dependent.

[0242] Table F: Antibodies in the presence or absence of CHO-K1-hFcγRIIb crosslinker cells

[0243] Stimulation of CD137-mediated signaling by CD137 antibodies

[0244]

[0245]

[0246] Example 8: Cross-linking-dependent activation of human primary B cells by masked anti-CD137 antibodies

[0247] The stimulatory activity of anti-CD137 antibodies and their shielded counterparts on CD137 receptor signaling was evaluated as follows. Human primary B cells were isolated from the peripheral blood of healthy donors. Jurkat-CD137-NFκB-luciferase reporter cells were cultured in the presence or absence of primary B cells as cross-linkers (E:CL=5:1 or 20:1). Serial dilutions of the test antibody were added to the reporter cell system to evaluate its ability to stimulate downstream luciferase activity. As shown in Figure 7, in the presence of human primary B cells as cross-linkers, at two E:CL ratios (5:1 or 20:1), the cleaved TY25368-MMP9 exhibited the strongest CD137 activation signal. The activity of the uncleaved shielded antibody TY25368 was much weaker than its cleaved form. Other clinical anti-CD137 antibodies were also compared in the test.

[0248] Example 9: Staphylococcal enterotoxin A (SEA) induces peripheral blood monocyte activation via masked anti-CD137 antibodies

[0249] Anti-CD137 antibodies were evaluated as follows to determine whether they could enhance the activation of human T cells stimulated by Staphylococcus enterotoxin A (SEA) peptide in the context of human peripheral blood mononuclear cells (PBMCs). SEA, a superantigen, is known to activate most human T cells by binding to MHC II expressed on the surface of antigen-presenting cells and T cell receptors (TCRs) expressed on the surface of T cells, and was therefore selected for initial T cell activation in this study. Human PBMCs (2.0×10 in 96-well plates) were isolated from two healthy donors (donor #102 and donor #142). 5 / well) and stimulated with suboptimal concentration (50 ng / mL) of SEA peptide. Next, serially diluted concentrations of anti-CD137 antibody and isotype control antibody were aliquoted into the wells. After 4 days, duplicate cell supernatants were collected and IL-2 was measured by ELISA as the endpoint for enhancing T cell activation.

[0250] As shown in Figure 8 and Table G, PBMCs from both donors expressed the highest levels of IL-2 cytokine secretion in the presence of cleaved TY25368 (i.e., TY25368 treated with MMP9). PBMCs from both donors had much weaker levels of IL-2 cytokine secretion in the presence of uncleaved TY25368. Other clinical anti-CD137 antibodies (e.g., AC1121 and AC1097) were also tested in this assay.

[0251] Table G: Enhancement of SEA-induced PBMC activation by anti-CD137 antibody

[0252]

[0253] Example 10: Screening of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) of Anti-CD137 Antibodies

[0254] Anti-CD137 antibodies and masked anti-CD137 antibodies were screened for antibody-dependent cell-mediated cytotoxicity (ADCC) activity as follows. Jurkat-CD16-NFAT-luciferase cells were co-cultured with 293-CD137 cells (i.e., 293F cells engineered to overexpress hCD137) as target cells. Serial dilutions of the test antibody were added to the ADCC reporter cell system to assess its ability to stimulate downstream luciferase activity. Fig. 9 As shown, in the presence of MMP9-treated TY25368, untreated TY25369, or IgG1 isotype control antibody, no ADCC reporter activity was detected on 293F-CD137 cells. In contrast, AC1121-IgG1 showed strong ADCC reporter activity on 293F-CD137 cells in a dose-dependent manner.

[0255] The complement dependent cytotoxicity (CDC) screening of the masked anti-CD137 antibody TY25368 is as follows. Human T cells isolated from healthy donors are activated by anti-CD3 / anti-CD28 to induce high levels of CD137 and HLA-A / B / C expression. The activated T cells are cultured in the presence of normal human serum complement (NHSC). Next, serially diluted test antibodies are added to the test system. Fig.10 As shown, no CDC activity was detected in the presence of MMP9-treated TY25368, untreated TY25368, or IgG1 isotype control antibody. In contrast, the positive control antibody (i.e., mouse anti-human HLA-A / B / C) showed strong CDC activity on activated T cells in a dose-dependent manner.

[0256] Example 11: Antitumor efficacy in the CT26 murine colon tumor model

[0257] BALB / c mice (female, 8 to 9 weeks old) were subcutaneously inoculated with CT26 mouse colon cancer cells. When the tumor volume reached approximately 80 mm 3 At the time of treatment, mice were randomly divided into four groups (n=8 per group) and treated with (a) vehicle control, (b) 1 mg / kg of shielded antibody TY25368, (c) 5 mg / kg of anti-PD-1 antibody FG1225, or (d) 1 mg / kg of shielded antibody TY25368 and 5 mg / kg of anti-PD-1 antibody FG1225. Treatment was intraperitoneal injection twice a week. Tumor growth was monitored twice a week, and the mean tumor volume ± SEM was reported over time. Fig.11As shown, TY25368 and FG1225 showed partial efficacy as single agents. On day 22 after the start of treatment, mice treated with TY25368 had a 48% inhibition of tumor growth, and mice treated with FC1225 had a 42% inhibition. In contrast, mice treated with a combination of TY25368 and FC1225 had an 85% inhibition of tumor growth.

[0258] Example 12: Antitumor efficacy in the MC38 murine colon tumor model

[0259] C57BL / 6 mice (female, 8 to 9 weeks old) were subcutaneously inoculated with MC38 mouse colon cancer cells. When the tumor volume reached approximately 80 mm 3 At 4 pm, mice were randomly divided into eight groups (n=8 per group) and treated with (a) vehicle, (b) 5 mg / kg of shielded antibody TY25368, (c) 0.1 mg / kg of anti-CTLA4 antibody TY21580, (d) 0.4 mg / kg of shielded anti-CTLA4 antibody TY22404, (e) 5 mg / kg TY25368 and 0.1 mg / kg TY21580, or (f) 5 mg / kg TY25368 and 0.4 mg / kg TY22404. Treatment was intraperitoneally injected twice a week. Tumor growth was monitored twice a week, and mean tumor volume ± SEM was reported over time. Fig.12 As shown, TY21580 and TY22404 showed partial efficacy as single agents. In the monotherapy group, 3 of 8 mice treated with TY21580 alone had tumor clearance. 1 of 8 mice treated with TY22404 alone had tumor growth inhibition. Combination treatment with TY25368 and TY21580, or TY25368 and TY22404, significantly enhanced the anti-tumor efficacy. See Fig.12 Five of the eight mice treated with TY25368 and TY21580 were tumor-free. All eight mice treated with TY25368 and TY22404 were tumor-free.

[0260] Example 13: Pharmacokinetic (PK) and pharmacodynamic (PD) studies of TY25368 in cynomolgus monkeys

[0261] Cynomolgus monkeys were intravenously administered 30 mg / kg TY25368 or 100 mg / kg TY25368 once a week for two weeks. The intact (i.e., uncleaved masked antibody) and total (i.e., uncleaved and cleaved forms) plasma concentrations of antibody TY25368 at different time points were measured using ELISA. The results are shown in Table H below. Peripheral T lymphocytes were also analyzed by FACS analysis, and no significant changes were observed (data not shown). The monkeys tolerated the antibody administration well, and no clinical symptoms were found during the study.

[0262] Table H: PK parameters of TY25368 in cynomolgus monkeys

[0263]

[0264] Cmax = maximum (peak) serum concentration; Tmax = time to reach Cmax; AUC = area under the curve describing the change in drug concentration in plasma over time; T1 / 2 = half-life; Vd = total systemic drug / drug plasma concentration; Cl = clearance.

[0265]

[0266]

[0267] like Fig.13 As shown in Table H, after the first dose, TY25368 showed a linear PK profile in both the total form and the intact form within the dose range between 30 mg / kg and 100 mg / kg. After the second 30 mg / kg dose, TY25368 exhibited rapid clearance and short T1 / 2. Avoiding theoretical constraints, this effect may be caused by treatment-induced anti-drug antibodies (ADA). Comparing the total form and the intact form after administration, highly similar drug concentrations and drug exposure were observed, indicating that the shielded antibody TY25368 was stable in the peripheral blood of monkeys. Therefore, preclinical toxicology studies indicate that TY25366 and TY25368 are well tolerated in monkeys with normal pharmacokinetic behavior and minimal circulation activation.

[0268] Example 14: Pharmacokinetic (PK) study of TY21242, TY24118 and TY25366 in mice

[0269] Pharmacokinetic studies were performed in BALB / c female mice bearing CT26 tumors. Mice were randomly divided into four groups (n=4 per group) and injected intraperitoneally with 5 mg / kg of TY25368, TY25366, TY21242, TY24118 or TY24122. Blood samples (approximately 50 μL / sample) were collected at 3, 6, 24, 48, 96, 168 and 336 hours after administration. The blood concentrations of TY25368, TY25366, TY21242, TY24118 and TY24122 were determined by ELISA using anti-human IgG Fc antibody as capture agent and HRP-labeled anti-human IgG (Fab specific) antibody as detection agent. A second set of ELISA assays were performed using specific anti-idiotypic antibodies as capture agents and HRP-labeled anti-human IgG (Fab-specific) antibodies as detection agents to detect the active (ie, unblocked) forms of TY25368 and TY25366. A description of each antibody is provided in Table 1.

[0270] Table I. Antibody Description

[0271]

[0272]

[0273] TY25368 has a half-life of 98 hours, and the drug concentration at 336 hours is about 6.48 μg / mL. TY25366 has a half-life of 28 hours, and the drug concentration at 336 hours is about 0.13 μg / mL. In comparison, the parent antibody TY21242 has a half-life of 74 hours, and the drug concentration at 336 hours is about 3.09 μg / ml; TY24118 has a half-life of 77 hours, and the drug concentration at 336 hours is about 2.50 μg / ml; TY24122 has a half-life of 79 hours, and the drug concentration at 336 hours is about 3.74 μg / ml. TY25368 has a slower clearance time and a longer half-life than the parent, while TY25366 has a much faster clearance time and a shorter half-life than the parent. No active forms of TY25366 or TY25368 were detected (data not shown), indicating that the masked antibodies were stable in the peripheral blood of mice.

[0274] Thus, preclinical toxicology studies indicated that TY25366 and TY25368 were well tolerated in mice with normal pharmacokinetic behavior and minimal circulating activation.

[0275] Example 15: Activation of CD137-mediated cell signaling by anti-CD137 antibodies containing Fc mutations

[0276] The Fc mutations in Table J, which may affect cross-linking, were introduced into TY23310 or TY21242 to generate TY24117, TY24118, TY24119, TY24120, TY24121, and TY24122.

[0277] Table J. Anti-CD137 Antibodies Comprising Fc Mutations

[0278]

[0279] a See Chu et al. (2008) Mol Immunol. 45(15):3926-33.

[0280] b See Mimoto et al. (2013) Protein Eng Des Sel. 26(10):589-98.

[0281] Anti-CD137 antibodies were transiently expressed in HEK293F cells and purified using standard protein A affinity chromatography (MabSelectSuRe, GE Healthcare). The agonistic activity of anti-CD137 antibodies was compared in a Jurkat / NFκB reporter gene assay. In brief, human embryonic kidney 293T cells were transiently transfected with a plasmid expressing the human CD137 receptor as well as a NFκB firefly luciferase reporter and a control Renilla luciferase reporter construct. The cells were incubated with the test antibodies in the presence or absence of (a) human CHO-K1-hFcγRIIb (also known as CHO-K1-FcγRIIb) or (b) mouse CHO-K1-mFcγRIIb. The relative level of CD137 signaling activation was then measured by firefly luciferase activity normalized by Renilla luciferase activity. Figures 14A to 14C As shown, in the presence of CHO-K1-FcγRIIb, both tested antibodies expressed higher CD137 agonistic activity compared to their wild-type IgG1 counterparts TY23310 or wild-type IgG4 counterparts TY21242. When CHO-K1-mFcγRIIb was used as a cross-linker, either no activity enhancement was detected or the activity enhancement detected was less than that observed in the human system. In the absence of a cross-linker, none of the antibodies exhibited CD137-mediated cell signaling.

[0282] Example 16: Staphylococcal enterotoxin A (SEA)-induced activation of peripheral blood mononuclear cells by anti-CD137 antibodies

[0283] The anti-CD137 antibodies generated in Example 15 were evaluated as follows to determine whether they could enhance the activation of human T cells stimulated by Staphylococcus enterotoxin A (SEA) peptide in the context of human peripheral blood mononuclear cells (PBMCs). Fresh human PBMCs were incubated for 96 hours with 50 ng / mL SEA and serially diluted concentrations of anti-CD137 antibodies (soluble form or immobilized on a solid support), and cell culture supernatants were subsequently collected to measure IL-2 levels. As shown in Figure 15, anti-CD137 antibodies TY24118 and TY24122 exhibited better agonistic activity than TY24117, TY24118, TY24119, TY24120, TY24121, and TY24122.

[0284] Example 17: Affinity of TY25368 and TY25368-MMP9 to human CD137, cynomolgus monkey CD137, mouse CD137, and rat CD137

[0285] The binding affinity of TY25368 and its activated form (called TY25368-MMP9) to recombinant CD137 from different species was evaluated by surface plasmon resonance (SPR). Briefly, anti-human IgG (Fc) antibody (Cytiva, catalog # BR-1008-39) was immobilized on a CM5 chip by amide coupling according to the instructions of the amine coupling kit (Cytiva, catalog # BR-1000-50). The final response of the fixed level was about 5000RU (relative units). TY25368 and TY25368-MMP9 were diluted to 3 μg / mL with 1×HBS-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA·2Na, 0.005% (v / v) Surfactant P20, pH 7.4), respectively, and then injected into the system at a flow rate of 10 μL / min for 30 seconds to fix. CD137 antigen was serially diluted with 1×HBS-EP buffer and flowed through the CM5 chip immobilized with the test antibody at a flow rate of 30 μL / min for 300 seconds. After association, 1×HBS-EP buffer was injected onto the surface at a flow rate of 30 μL / min for 300 seconds to test the antigen dissociation rate. 3M magnesium chloride solution was used to remove residual antibody / antigen for regeneration of the CM5 sensor chip. As shown in Table K, TY25368 has low binding affinity (KD>1000nM) for human CD137, cynomolgus monkey CD137, mouse CD137, and rat CD137. In contrast, activated TY25368-MMP9 was found to bind to human CD137 and cynomolgus monkey CD137 with high affinity (KD of 3.19 and 4.23nM, respectively). Activated TY25368-MMP9 was also found to bind to mouse CD137 and rat CD137 with lower affinity (KD of 27.54 and 42.37 nM, respectively).

[0286] Table K: Affinity of TY25368 and TY25368-MMP9 to CD137 from different species measured by SPR.

[0287]

[0288]

[0289] Example 18: Anti-tumor efficacy of TY25368 in the mouse EMT6 breast cancer model

[0290] BALB / c mice (4 groups, n=8 per group, female, 8 to 9 weeks old) were subcutaneously inoculated with EMT6 (ATCC) mouse breast cancer cells. When tumors were established (i.e., when the tumor volume reached approximately 120 mm 3Mice were treated with (a) vehicle, (b) 3 mg / kg of shielded anti-CD137 antibody TY25368, (c) 1 mg / kg TY25368, or (d) 0.3 mg / kg TY25368 at 4 pm. Treatment was intraperitoneal injection twice a week. Tumor growth was monitored twice a week, and mean tumor volume ± SEM was reported over time. Fig.16 As shown, TY25368 demonstrated a dose-dependent antitumor effect in the EMT6 mouse allograft tumor model. Tumor regression was observed in all mice treated with 1 mg / kg TY25368 and 3 mg / kg TY25368.

[0291] Example 19: Antitumor efficacy of TY24118, TY24122, TY25366 and TY25368 in the H22 mouse liver cancer model

[0292] BALB / c mice (5 groups, n=8 per group, female, 8 to 9 weeks old) were subcutaneously inoculated with H22 (CCTCC) mouse hepatoma cells. When tumors were established (i.e., when the tumor volume reached approximately 90 mm 3 Mice were treated with (a) vehicle, (b) 5 mg / kg TY24118, (c) 5 mg / kg TY24122, (d) 5 mg / kg TY25366, or (e) 5 mg / kg TY25368 (Table I above provides descriptions of the antibodies). Treatments were given by intraperitoneal injection twice a week. Tumor growth was monitored twice a week, and mean tumor volumes ± SEM are reported over time. Fig.17 As shown, parental anti-CD137 antibodies TY24118 and TY24122 (unshielded), and shielded anti-CD137 antibodies TY25366 and TY25368 all exhibited similar efficacy at 5 mg / kg.

[0293] Example 20: Antitumor efficacy of TY24118, TY24122, TY25366 and TY25368 in the mouse CT26 colorectal cancer model

[0294] BALB / c mice (5 groups, n=8 per group, female, 9 to 10 weeks old) were subcutaneously inoculated with CT26 (SIBS) mouse colon cancer cells. When tumors were established (i.e., when the tumor volume reached approximately 60 mm 3 Mice were treated with (a) vehicle, (b) TY24118, (c) TY24122, (d) TY25366, or (e) TY25368 (Table I above provides descriptions of the antibodies). Treatments were administered ip twice a week at 5 mg / kg or 1 mg / kg of antibody. Treatments were administered ip twice a week. Tumor growth was monitored twice a week and mean tumor volume ± SEM is reported over time. Fig.18Aand 18B As shown, the parental anti-CD137 antibodies TY24118 and TY24122 (unshielded) showed similar efficacy at both high doses (5 mg / kg) and low doses (1 mg / kg). The shielded anti-CD137 antibody TY25368 showed similar efficacy to TY24118 and TY24122 at both high doses (5 mg / kg) and low doses (1 mg / kg). The shielded anti-CD137 antibody TY25366 was not as effective as TY24118 and TY24122 at both high doses (5 mg / kg) and low doses (1 mg / kg).

[0295] Exemplary conclusions

[0296] TY25368 is an Fc-enhanced, shielded anti-CD137 with broad species cross-reactivity. TY25368 has high shielding efficiency and is conditionally activated to strongly bind to the CD137 co-stimulatory receptor on activated T cells. TY25368 shows FcγR-dependent stimulation of strong CD137 signaling, which is more effective than urelumab. In the presence of a primary stimulation signal, TY25368 exhibits stronger anti-CD137 agonist activity than urelumab on T cell activation, while shielded TY25368 has much lower activity. TY25368 has strong anti-tumor activity as a single agent and mediates enhanced anti-tumor efficacy in combination with other immune checkpoint inhibitors, including anti-PD-1 or anti-CTLA-4. In nonclinical toxicology studies, TY25368 was well tolerated in rats and cynomolgus monkeys with normal pharmacokinetic behavior and minimal circulating activation.

[0297] Example 21: Antitumor efficacy of the combination of TY25368 and TY27151 in the mouse MC38-HER2-B7H3 colon cancer model

[0298] C57BL / 6-hCD3e mice (n=6 / group, female, 9 weeks old) were inoculated subcutaneously with MC38-HER2-B7H3 mouse colon cancer cells. C57BL / 6-hCD3e mice are derived from C57BL / 6 with a human CD3E knock-in gene. These mice express hCD3E on their T cells. The MC38-HER2-B7H3 cell line is derived from the MC38 cell line and is engineered to overexpress human HER2 ("hHER2") and human B7H3 ("hB7H3"). When tumors are established (i.e., when the tumor volume reaches approximately 105 mm 3Mice were treated with (a) vehicle, (b) TY25368, (c) TY27151, or (d) TY25368 and TY27151. 5 mg / kg of the antibody was administered twice a week by intraperitoneal injection. Table I above provides a description of TY25368. TY27151 is a bispecific T cell engager (TCE) construct that binds HER2 and CD3. The anti-CD3 and anti-HER2 arms of TY27151 each contain a shielding portion and a linker unit, which contains a protease cleavage site. The heavy and light chain amino acid sequences of TY27151 are shown in Table L below. The shielding peptide of TY27151 is in bold. The shielding sequence is underlined and the linker unit is italicized. TY27151 comprises three polypeptide chains: an anti-HER2 antibody heavy chain and an anti-HER2 antibody light chain, and a ScFv-Fc fusion polypeptide, wherein the C-terminus of the ScFv is fused to the Fc domain to the N-terminus. The anti-HER2 antibody heavy chain and the anti-HER2 antibody light chain associate to form an anti-HER2 binding arm. The ScFv-Fc fusion polypeptide binds to CD3. The Fc domain of the anti-HER2 binding arm and the Fc of the anti-CD3 ScFv-Fc fusion polypeptide dimerize to form a TCE. For example, see Fig. 20 Schematic diagram of TY27151 in . Additional details about TY27151 are provided in WO 2022 / 170740, which is incorporated herein by reference in its entirety.

[0299] Table L: Heavy and light chain amino acid sequences of TY27151

[0300]

[0301]

[0302] *Obscissa sequences are bold underlined; connecting units are bold italic

[0303] When tumors were established, treatment was initiated by intraperitoneal injection twice a week with vehicle, TY25368 (5 mg / kg), HER2xCD3 dual shielded bispecific antibody TY27151 (5 mg / kg), or a combination thereof. Tumor growth was monitored twice a week, and mean tumor volume ± SEM was reported over time. Fig.19A As shown in Figure 2, TY25368 showed very weak efficacy as a single agent, with a TGI of 19% on day 22. TY27151 showed moderate efficacy as a single agent, with a TGI of 54% on day 22 and 2 / 8 mice cleared of tumor. Combination therapy of TY25368 and TY27151 significantly enhanced the anti-tumor efficacy, with a TGI of 85% on day 22 and 3 / 8 mice cleared of tumor. Tumor growth curves of individual mice in each treatment group (a), (b), (c) and (d) are shown in Figure 2. Fig.19B shown.

[0304] The present invention has been described in accordance with the specific embodiments comprising the preferred modes for practicing the present invention discovered or proposed by the inventors. Those skilled in the art will appreciate that, in view of the present disclosure, various modifications and changes may be made to the illustrated specific embodiments without departing from the intended scope of the present invention. For example, due to codon redundancy, changes may be made to the potential DNA sequence without affecting the protein sequence. In addition, due to considerations of biological functional equivalence, changes may be made to the protein structure without affecting the type or quantity of the biological action. All such modifications are intended to be included within the scope of the accompanying patent.

Claims

1. A shielded antibody comprising a shielding peptide (MP) and an antibody that binds to human CD137, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL); wherein the MP is linked to the N-terminus of the VL, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus, and wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7; and wherein the VH comprises the CDR-H1 shown in TGGVGVG (SEQ ID NO:36), the CDR-H2 shown in LIDWADDKYYSPSLKS (SEQ ID NO:37), and the CDR-H3 shown in GGSDTVIGDWFAY (SEQ ID NO:38), and wherein the VL comprises the CDR-L1 shown in RASQSIGSYLA (SEQ ID NO:39), the CDR-L2 shown in DASNLET (SEQ ID NO:40), and the CDR-L3 shown in QQGYYLWT (SEQ ID NO:41).

2. The shielded antibody of claim 1, wherein the MP further comprises an N-terminal unit (NU) linked to the N-terminus of the MU.

3. The shielded antibody of claim 2, wherein the N-terminal unit is about 1-10 amino acid residues in length.

4. The shielded antibody of claim 3, wherein the N-terminal unit comprises E or EVGSY (SEQ ID NO: 77).

5. The shielded antibody of any one of claims 1 to 4, wherein the LU comprises a first cleavage site.

6. The shielded antibody of claim 5, wherein the first cleavage site is selected from the group consisting of protease cleavage sites of the following proteases: urokinase-type plasminogen activator / uPA, matrix metalloproteinase-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus protease / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Casparase-1, Casparase-2, Casparase-3, Casparase-4, Casparase-5, Casparase-6, Casparase-7, Casparase-8, Casparase-9, Casparase-10, Casparase-11, Casparase-12, Casparase-13, Casparase-14 and TACE.

7. The masked antibody of claim 5, wherein the first cleavage site is a protease cleavage site of urokinase-type plasminogen activator / uPA or MMP-9.

8. The shielded antibody of claim 5 or 6, wherein the LU further comprises a first linker (L1).

9. The shielded antibody of any one of claims 5 to 7, wherein the LU further comprises a second cleavage site.

10. The shielded antibody of claim 8, wherein the second cleavage site is at the C-terminus of the L1.

11. The shielded antibody of claim 9 or 10, wherein the second cleavage site is selected from the group consisting of protease cleavage sites of the following proteases: urokinase-type plasminogen activator / uPA, matrix metalloproteinase-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus protease / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Casparase-1, Casparase-2, Casparase-3, Casparase-4, Casparase-5, Casparase-6, Casparase-7, Casparase-8, Casparase-9, Casparase-10, Casparase-11, Casparase-12, Casparase-13, Casparase-14 and TACE.

12. The masked antibody of claim 11, wherein the second cleavage site is a protease cleavage site of urokinase-type plasminogen activator / uPA or MMP-9.

13. The masked antibody of any one of claims 9 to 12, wherein the first cleavage site and the second cleavage site are the same.

14. The masked antibody of any one of claims 9 to 12, wherein the first cleavage site and the second cleavage site are different.

15. The shielded antibody of any one of claims 10 to 14, wherein the LU further comprises a second linker (L2).

16. The masked antibody of any one of claims 5 to 15, wherein the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-16.

17. The shielded antibody of any one of claims 1 to 16, wherein the shielding peptide comprises any one of SEQ ID NOs: 17-35.

18. The shielded antibody of claim 17, wherein the shielding peptide comprises SEQ ID NO:

34.

19. The masked antibody of any one of claims 1 to 18, wherein the antibody comprises the VH shown in SEQ ID NO: 52 and the VL shown in SEQ ID NO:

53.

20. The masked antibody of claims 18 and 19, wherein the VH comprises SEQ ID NO: 52 and the VL comprises SEQ ID NO:

58.

21. The masked antibody of any one of claims 1 to 20, wherein the masked antibody is a full length antibody comprising an Fc region.

22. The shielded antibody of claim 21, wherein the Fc region is a human IgG Fc region or a variant thereof.

23. The shielded antibody of claim 22, wherein the human IgG Fc region or a variant thereof is a human IgG1 Fc region, a human IgG2 Fc region, a human IgG4 Fc region, or a variant of any of the foregoing.

24. The shielded antibody of claim 23, comprising a protein selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and and A330R; G237D, H268D, P238D and P271G; G237D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and E233D, G237D, P238D, H268D, P271G and A330R; S2657A; T437R; K248E; and human IgG1 Fc region variants consisting of a group of substitutions, wherein the amino acid numbering is according to the EU index.

25. The masked antibody of claim 24, comprising a human IgG1 Fc region variant comprising S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index.

26. The masked antibody of claim 25, comprising an Fc region comprising SEQ ID NO: 113 or SEQ ID NO:

114.

27. The masked antibody of any one of claims 18 to 26, comprising the masking peptide shown in SEQ ID NO:34, the antibody heavy chain variable region shown in SEQ ID NO:52 and the antibody light chain variable region shown in SEQ ID NO:53, and a human IgG1 Fc region variant comprising S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index.

28. The masked antibody of claim 27, comprising a heavy chain comprising SEQ ID NO: 94 or 95 and a light chain comprising SEQ ID NO:

96.

29. The shielded antibody of claim 23, comprising a protein selected from the group consisting of G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D and A330R; E233D, P231G, P238D and A330R; G237D, H268D, P238D and P271G; G237D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and E233D, G237D, P238D, H268D, P271G and A330R; S2657A; T437R; K248E; and human IgG4 Fc region variants consisting of a group of substitutions, wherein the amino acid numbering is according to the EU index.

30. The masked antibody of claim 29, comprising a human IgG4 Fc region variant comprising S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index.

31. The shielded antibody of claim 30, comprising an Fc region comprising SEQ ID NO: 117 or 118.

32. The masked antibody of any one of claims 18 to 23 and 29 to 31, comprising the masking peptide of SEQ ID NO: 34, the antibody heavy chain variable region shown in SEQ ID NO: 52 and the antibody light chain variable region shown in SEQ ID NO: 53, and a human IgG4 Fc region variant comprising S267E and L328F substitutions, wherein the amino acid numbering is according to the EU index.

33. The masked antibody of claim 32, comprising a heavy chain comprising SEQ ID NO: 92 or 93 and a light chain comprising SEQ ID NO:

96.

34. The shielded antibody of any one of claims 1 to 20, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, (scFv)2, linear antibodies, single-chain antibodies, single-domain antibodies (nanobodies), VHH, minibodies, or diabodies.

35. One or more polynucleotides encoding the masked antibody of any one of claims 1 to 34.

36. A recombinant vector comprising one or more polynucleotides according to claim 35.

37. A host cell comprising the vector of claim 36.

38. A method of producing a masked antibody, comprising culturing the host cell as claimed in claim 37 under suitable conditions to cause expression of the masked antibody and recovering the masked antibody.

39. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the shielded antibody of any one of claims 1 to 34.

40. The method of claim 39, wherein the cancer is a solid tumor cancer.

41. The method of claim 40, wherein the solid tumor is breast cancer, liver cancer, colorectal cancer, or colon cancer.

42. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a shielded antibody according to any one of claims 1 to 34 and an effective amount of an anti-PD-1 antibody.

43. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a shielded antibody according to any one of claims 1 to 34 and an effective amount of an anti-CTLA-4 antibody.

44. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a shielded antibody as described in any one of claims 1 to 34 and an effective amount of a bispecific antibody that binds HER2 and CD3.

45. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a shielded antibody as described in any one of claims 1 to 34 and an effective amount of a bispecific T cell engager (TCE) targeting CD3 and an antigen expressed on the surface of a solid tumor cancer cell.

46. ​​The method of claim 45, wherein the antigen expressed on the surface of the solid tumor cancer cells is HER2.

47. A method as described in claim 45 or 46, wherein the TCE comprises three polypeptide chains, wherein the first polypeptide chain comprises an antibody heavy chain, the second polypeptide chain comprises an antibody light chain, and the third polypeptide chain comprises an scFv-Fc, wherein the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, wherein the first and second polypeptide chains associate to form a HER2 binding arm, wherein the third polypeptide chain binds to CD3, and wherein the Fc domain of the anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form the TCE.

48. The method of claim 47, wherein the first polypeptide chain comprises SEQ ID NO: 125, the second polypeptide chain comprises SEQ ID NO: 124, and the third polypeptide chain comprises SEQ ID NO:

126.

49. The method of any one of claims 42 to 47, wherein the cancer is a solid tumor.

50. The method of claim 48, wherein the solid tumor is colon cancer, breast cancer, liver cancer, colorectal cancer, or colon cancer.

51. A kit comprising a shielded antibody as claimed in any one of claims 1 to 34 for use in treating an individual suffering from cancer according to the method as claimed in any one of claims 39 to 41.

52. A kit comprising a masked antibody as claimed in any one of claims 1 to 34 for use in combination with an anti-PD-1 antibody to treat an individual suffering from cancer according to the method of any one of claims 42 and 46 to 47.

53. A kit comprising a masked antibody as claimed in any one of claims 1 to 36 for use in combination with an anti-CTLA4 antibody to treat an individual suffering from cancer according to the method of any one of claims 43 and 48 to 49.

54. A kit comprising a masked antibody as claimed in any one of claims 1 to 36 for use in treating an individual suffering from cancer in combination with a bispecific antibody that binds HER2 and CD3 according to the method of any one of claims 44 and 48 to 49.

55. A kit comprising a masked antibody as described in any one of claims 1 to 36 for use in treating an individual with cancer according to the method of any one of claims 45 to 49 in combination with a bispecific T cell engager targeting CD3 and an antigen expressed on the surface of a solid tumor cancer cell.

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