Anti-CD137 antibodies and methods for making and using same

Abstract

Provided herein is a masked anti-CD137 antibody, a nucleic acid encoding such an antibody, a vector comprising such a nucleic acid, and a host cell comprising such a nucleic acid or vector.Also provided is a method for producing masked anti-CD137 antibody.Further provided is a method for treating cancer, comprising administering an effective amount of masked anti-CD137 antibody as a monotherapy or in combination with another therapeutic antibody.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 079475, filed March 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (695402002241SEQLIST.xml, size: 126,181 bytes, creation date: March 3, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to masked anti-CD137 antibodies and antigen-binding fragments thereof that bind to human CD137, compositions comprising same, and uses thereof to delay and / or prevent tumor growth. [Background technology]

[0004] CD137 (also called CD137 receptor, 4-1BB, TNFRSF9, etc.) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). Current understanding of CD137 indicates that its expression is generally activation-dependent and is present on a broad subset of immune cells, including activated NK and NKT cells, regulatory T cells, dendritic cells (DCs), stimulatory mast cells, differentiated myeloid cells, monocytes, neutrophils, and eosinophils (Wang, 2009, Immunological Reviews 229:192-215). Expression of CD137 has also been demonstrated on tumor vasculature (Broll, 2001, Amer. J. Clin. Pathol. 115(4):543-549; Seaman, 2007, Cancer Cell 11:539-554) and at sites of inflammation or atherosclerosis endothelium (Drenkard, 2007 FASEB J. 21:456-463; Olofsson, 2008, Circulation 117:1292-1301). The stimulatory ligand for CD137, CD137 ligand (CD137L), is expressed on activated antigen-presenting cells (APCs), myeloid progenitor cells, and hematopoietic stem cells.

[0005] Human CD137 is a 255 amino acid protein (GenBank Accession Nos. NM_001561; NP_001552; SEQ ID NO: 1). The protein contains 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 dimeric forms and can trimerize with CD137 ligand to transmit signals.

[0006] Numerous studies of mouse and human T cells have shown that CD137 promotes increased cell proliferation, survival, and cytokine production (Croft, 2009, Nat Rev Immunol 9:271-285). Studies have shown that several CD137 agonist monoclonal antibodies (mAbs) increase the expression of costimulatory molecules, significantly enhance cytolytic T lymphocyte responses, and provide antitumor effects in a variety of models. CD137 agonist mAbs have demonstrated efficacy in preventative and therapeutic settings. Furthermore, CD137 monotherapy and combination therapy tumor models have established durable antitumor 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 offers the potential to provide antitumor activity while limiting the autoimmune side effects that may be associated with immunotherapeutic approaches that break immune tolerance.

[0007] However, liver-associated autoimmune toxicity caused by agonistic anti-CD137 antibodies has significantly limited their use in clinical applications.There has been a long unmet need for antibodies that bind to human CD137, enhance CD137-mediated responses, and provide potential therapeutic agents for treating a variety of diseases and conditions (e.g., cancer) without immune-mediated side effects. Summary of the Invention

[0008] In some embodiments, provided herein is a masked antibody comprising a masking 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, and the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), and the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7; wherein the VH comprises CDR-H1 represented by TGGVGVG (SEQ ID NO: 36), CDR-H2 represented by LIDWADDKYYSPSLKS (SEQ ID NO: 37), and CDR-H3 represented by GGSDTVIGDWFAY (SEQ ID NO: 38), and the VL comprises CDR-L1 represented by RASQSIGSYLA (SEQ ID NO: 39), CDR-L2 represented by DASNLET (SEQ ID NO: 40), and CDR-L3 represented by QQGYYLWT (SEQ ID NO: 41). In some embodiments, the MP further comprises an N-terminal unit (NU) linked to the N-terminus of said MU. In some embodiments, the N-terminal unit is about 1-10 amino acid residues in length. In some embodiments, the N-terminal unit comprises E or EVGSY (SEQ ID NO: 77).

[0009] In some embodiments, the LU comprises the first cleavage site. In some embodiments, the first cleavage site is a protease cleavage site for a protease selected from the group consisting of urokinase-type plasminogen activator / uPA, matrix metalloproteinase-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first cleavage site is a urokinase-type plasminogen activator / uPA or MMP-9 protease cleavage site. In some embodiments, the LU further comprises a first linker (L1) C-terminal to the first cleavage site. In some embodiments, the LU further comprises a second cleavage site. In some embodiments, the second cleavage site is C-terminal to L1. In some embodiments, the second cleavage site is a protease cleavage site for a protease selected from the group consisting of urokinase-type plasminogen activator / uPA, matrix metalloproteinase-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the second cleavage site is a protease cleavage site for urokinase-type plasminogen activator / uPA or MMP-9. In some embodiments, the first and said second cleavage sites are the same.In some embodiments, the first and second cleavage sites are different. In some embodiments, the LU further comprises a second linker (L2) C-terminal to the second cleavage site. In some embodiments, the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-16.

[0010] In some embodiments, the masking peptide comprises any one of SEQ ID NOs: 17-35. In some embodiments, the masking peptide comprises SEQ ID NO: 34. In some embodiments, the antibody comprises a VH set forth in SEQ ID NO: 52 and a VL set forth in SEQ ID NO: 53. In some embodiments, the VH comprises SEQ ID NO: 52 and the VL comprises SEQ ID NO: 58.

[0011] 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 above. In some embodiments, the masked antibody comprises mutants of a human IgG1 Fc region, the mutants of the human IgG1 Fc region being 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 T437R and K248E, where amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a variant of a human IgG1 Fc region, the variant of the human IgG1 Fc region comprising S267E and L328F substitutions, amino acid numbering according to the EU index. In some embodiments, the masked antibody comprises said Fc region comprising SEQ ID NO:113 or SEQ ID NO:114.

[0012] In some embodiments, the masked antibody comprises the masking peptide set forth in SEQ ID NO: 34, the antibody heavy chain variable domain set forth in SEQ ID NO: 52, and the antibody light chain variable domain set forth in SEQ ID NO: 53, and the human IgG1 Fc region variant comprising S267E and L328F substitutions, where amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises the heavy chain comprising SEQ ID NO: 94 or 95, and the light chain comprising SEQ ID NO: 96. In some embodiments, the masked antibody comprises mutants of a human IgG4 Fc region, the mutants of the human IgG4 Fc region being 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 T437R and K248E, where amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a variant of a human IgG4 Fc region, the variant of the human IgG4 Fc region comprising an S267E and L328F substitution, where the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises said Fc region comprising SEQ ID NO: 117 or 118. In some embodiments, the masked antibody comprises said masking peptide set forth in SEQ ID NO: 34, said antibody heavy chain variable domain set forth in SEQ ID NO: 52 and said antibody light chain variable domain set forth in SEQ ID NO: 53, and said human IgG4 Fc region variant comprising an S267E and L328F substitution, where the amino acid numbering is according to the EU index.In some embodiments, the masked antibody comprises the heavy chain comprising SEQ ID NO:92 or 93 and the light chain comprising SEQ ID NO:96.

[0013] In some embodiments, the masked antibody is a masked antibody fragment selected from the group consisting of a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, (scFv)2, a linear antibody, a single chain antibody, a single domain antibody (nanobody) VHH, a minibody, or a diabody.

[0014] In some embodiments, one or more polynucleotides are provided that encode the masked antibodies described herein. In some embodiments, a recombinant vector is provided that includes one or more polynucleotides described herein. In some embodiments, a host cell is provided that includes a vector described herein. In some embodiments, a method of producing a masked antibody is provided, the method comprising culturing a host cell described herein under suitable conditions to cause expression of the masked antibody and recovering the masked antibody.

[0015] In some embodiments, a method of treating cancer in a subject is provided, the method comprising administering to the subject an effective amount of a masked antibody provided herein. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor is breast cancer, liver cancer, colon cancer, or colon cancer.

[0016] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of a masked antibody 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 of treating cancer in a subject is provided, comprising administering to the subject an effective amount of a masked antibody described herein and an effective amount of an anti-CTLA4 antibody (e.g., an anti-human CTLA4 antibody). In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of a masked antibody 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 of treating cancer in a subject is provided, comprising administering to the subject an effective amount of a masked antibody described herein and an effective amount of a bispecific T cell engager (TCE) that targets 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, 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 domain fusion, the C-terminus of the scFv is fused to the N-terminus of the Fc domain, the first and second polypeptide chains form an associated HER-2 binding arm, the third polypeptide chain binds to CD3, and 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 masked TCE (e.g., the portion of the TCE that binds to CD3 and / or the portion of the TCE that binds to HER2 is masked). 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, colon cancer, or colon cancer.

[0017] In some embodiments, a kit is provided that includes a masked antibody described herein for treating an individual with cancer according to the methods described herein. In some embodiments, a kit is provided that includes a masked antibody described herein for use 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 is provided that includes a masked antibody described herein for use 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 is provided that includes a masked antibody described herein for use 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 is provided that includes a masked antibody described herein for use in combination with a bispecific T cell engager (TCE) that targets an antigen expressed on the surface of a solid tumor cancer cell and CD3 (e.g., human CD3) 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 that targets CD3 and HER2 comprises three polypeptide chains, a first polypeptide chain comprises an antibody heavy chain, a second polypeptide chain comprises an antibody light chain, and a third polypeptide chain comprises an scFv-Fc domain fusion, where the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, the first and second polypeptide chains associate to form a HER-2 binding arm, and the third polypeptide chain binds to CD3, and the domain of the Fc anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form the TCE. In some embodiments, the TCE is a masked TCE (e.g., the portion of the TCE that binds to CD3 and / or the portion of the TCE that binds to HER2 is masked).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 characteristics of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be 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]

[0018] [Figure 1A] FIG. 1B shows the results of a FACS-based assay performed to evaluate the masking efficiency of an exemplary CD137-masked antibody, as compared to the parental antibodies TY21242 and TY23310, against human CD137 displayed on the surface of yeast cells. FIG. 1B also shows the results of a FACS-based assay performed to evaluate the masking efficiency of an exemplary CD137-masked antibody, as compared to the parental antibodies TY21242 and TY23310, against human CD137 displayed on the surface of yeast cells. FIG. 1C shows additional results of a FACS-based assay performed to evaluate the masking efficiency of an exemplary CD137-masked antibody, as compared to the parental antibodies TY21242 and TY23310, against human CD137 displayed on the surface of yeast cells. FIG. 1D shows additional results of a FACS-based assay performed to evaluate the masking efficiency of an exemplary CD137-masked antibody, as compared to the parental antibodies TY21242 and TY23310, against human CD137 displayed on the surface of yeast cells. [Figure 2A] shows the results of an ELISA assay performed to evaluate the activity of the masked anti-CD137 antibodies TY25366 and TY25368 before removal of the masking peptide (i.e., without MMP9 treatment) and after removal of the masking peptide (i.e., with MMP9 treatment) in comparison to the parental antibodies TY24118 and TY24122, respectively. See also Table D. [Figure 2B] shows the results of a FACS-based assay performed to evaluate the activity of masked anti-CD137 antibodies TY25366 and TY25368 compared to the parental antibodies TY24118 and TY24122, respectively, before removal of the masking peptide (i.e., without MMP9 treatment) and after removal of the masking peptide (i.e., with MMP9 treatment). See also Table D. [Figure 3A-3E] Figure 3A(i) shows the size exclusion chromatography (SEC) profiles of exemplary masked antibodies TY25366 and TY25368 under accelerated stress conditions. Figure 3A(i) shows the SEC profile of TY25366 after 0, 3, and 6 freeze-thaw cycles. Figure 3A(ii) shows the SEC profile of TY25368 after 0, 3, and 6 freeze-thaw cycles. Figure 3B(i) shows the SEC profile of TY25366 after storage at 40 °C for 0, 7, 14, 21, and 28 days. Figure 3B(ii) shows the SEC profile of TY25368 after storage at 40 °C for 0, 7, 14, 21, and 28 days. Figure 3C(i) shows the SEC profile of TY25366 after storage at room temperature in acidic buffer (sodium acetate solution, pH 3.6) for 0 and 2 hours. Figure 3C(ii) shows the SEC profile 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 at 40 °C for 0 and 24 hours in 50 mM histidine, 300 mM NaCl, pH 7.0 buffer. Figure 3D(ii) shows the SEC profile of TY25368 after storage at 40 °C for 0 and 24 hours in 50 mM histidine, 300 mM NaCl, pH 7.0 buffer. Figure 3E(i) shows the SEC profiles of TY25366 and TY25368 before and after storage in saline at room temperature for 6 hours and at 4 °C for 24 hours compared to the control condition. [Figure 4A(i)]FIG. 4A(ii) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD4+ human T cells. FIG. 4A(ii) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD8+ human T cells. FIG. 4B(i) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD4+ cynomolgus monkey T cells. FIG. 4B(ii) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD8+ cynomolgus monkey T cells. FIG. 4C(i) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD4+ mouse T cells. FIG. 4C(ii) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD8+ mouse T cells. FIG. 4D(i) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD4+ rat T cells. FIG. 4D(ii) shows the results of a FACS-based assay performed to evaluate the binding of MMP9-treated TY25368 and untreated TY25368 to naive and activated CD8+ rat T cells. [Diagram 5] shows the results of ELISA experiments performed to assess the extent to which MMP9-treated and untreated TY25368 inhibits the binding of CD137 to its ligands. [Figure 6A]Figure 6B shows the results of an experiment performed to assess the extent to which various masked and unmasked anti-CD137 antibodies stimulate CD137 signaling. Signaling via CD137 was determined using a CD137 reporter gene assay in the presence of CHO-K1-hFcgRIIb cells (E:CL = 20:1) as a crosslinker. Figure 6C shows the results of an experiment performed to assess the extent to which various masked and unmasked anti-CD137 antibodies stimulate CD137 signaling. Signaling via CD137 was determined using a CD137 reporter gene assay in the absence of CHO-K1-hFcgRIIb cells as a crosslinker. [Figure 7A] Figure 7B shows the results of an experiment performed to assess the extent to which different masked and unmasked anti-CD137 antibodies stimulate CD137 signaling. Signaling via CD137 was determined using a CD137 reporter gene assay in the presence of human primary B cells (E:CL=5:1) as a crosslinker. Figure 7C shows the results of an experiment performed to assess the extent to which different masked and unmasked anti-CD137 antibodies stimulate CD137 signaling. Signaling via CD137 was determined using a CD137 reporter gene assay in the presence of human primary B cells (E:CL=20:1) as a crosslinker. [Figure 8A] shows the results of an ELISA-based assay performed to determine whether anti-CD137 antibodies promote SEA-stimulated cytokine secretion by human PBMCs obtained from donor #102. Figure 8B shows the results of an ELISA-based assay performed to determine whether anti-CD137 antibodies promote SEA-stimulated cytokine secretion by human PBMCs obtained from donor #142. [Figure 9] shows the results of experiments performed to evaluate the ADCC activity of anti-CD137 and masked anti-CD137 antibodies. [Figure 10] presents the results of experiments performed to evaluate the CDC activity of masked anti-CD137 antibodies. [Figure 11] shows the results of in vivo experiments performed to evaluate the efficacy of (a) vehicle, (b) masked anti-CD137 antibody TY25368, (c) anti-PD-1 antibody FG1225, and (d) TY25368 in combination with FG1225 to inhibit the growth of CT26 murine colon tumors in a mouse allograft model. [Figure 12] shows the results of in vivo experiments performed to evaluate the efficacy of (a) vehicle, (b) masked anti-CD137 antibody TY25368, (c) masked anti-CTLA4 antibody TY21580, (d) masked anti-CTLA4 antibody TY22404, (e) masked anti-CD137 antibody TY25368 and masked anti-CTLA4 antibody TY21580, and (f) masked anti-CD137 antibody TY25368 and masked anti-CTLA4 antibody TY22404 in inhibiting the growth of MC28 mouse colon tumors in a mouse allograft model. [Figure 13] shows the concentration-time profiles of intact and total forms of TY25366 and TY25368 in cynomolgus monkeys at doses of 30 and 100 mg / kg. [Figure 14A] Figure 14A shows the results of an experiment 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. Figure 14B shows the results of an experiment performed 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. Figure 14C shows the results of an experiment performed to evaluate the activation of CD137-mediated cell signaling by anti-CD137 antibodies containing Fc mutations in the absence of a cross-linking agent. [Figure 15A]Figure 15B shows the results of an experiment performed to assess the extent to which an anti-CD137 antibody containing an Fc mutation immobilized on a solid support enhances SEA-stimulated cytokine secretion by human PBMCs. Figure 15B shows the results of an experiment performed to assess the extent to which a soluble anti-CD137 antibody containing an Fc mutation enhances SEA-stimulated cytokine secretion by human PBMCs. [Figure 16] shows the results of in vivo experiments conducted to evaluate the efficacy of (a) vehicle, (b) 3 mg / kg masked anti-CD137 antibody TY25368, (c) 1 mg / kg masked anti-CD137 antibody TY25368, and (d) 0.3 mg / kg masked anti-CD137 antibody TY25368 in inhibiting the growth of EMT6 mouse breast cancer tumors in a mouse allograft model. [Figure 17] shows 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 H22 mouse hepatocarcinoma tumors in a mouse allograft model. [Figure 18A] Figure 18B shows the results of an in vivo experiment 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 to inhibit the growth of CT26 mouse colon carcinoma tumors in a mouse allograft model. Figure 18C shows the results of an in vivo experiment 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 to inhibit the growth of CT26 mouse colon carcinoma tumors in a mouse allograft model. [Figure 19A]shows 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 mouse colon carcinoma tumors expressing human HER2 and human B7H3 in a mouse syngeneic tumor model. Figure 19B shows the tumor growth curves for individual mice in each treatment group (a), (b), (c), and (d). [Figure 20] FIG. 1 is a schematic diagram of TY27151. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] overview Toxicity resulting from systemic stimulation of T cells limits the application of many CD137 agonists in cancer immunotherapy. The present invention provides a masked anti-CD137 antibody that is effective in treating cancer and does not pose significant safety issues.

[0020] definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0021] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and so forth.

[0022] As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0023] It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0024] As used herein, the term "and / or" is intended to mean that phrases such as "A and / or B" include both A and B, A or B, A (single), and B (single). Similarly, as used herein, the term "and / or" is intended to mean that phrases such as "A, B, and / or C" include 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 (single); B (single); and C (single).

[0025] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

[0026] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can 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. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can contain modification(s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps" which replace one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, 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 intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or replaced with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0027] 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 that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes a nucleic acid molecule that is separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid of interest).

[0028] The term "antibody" is used herein in the broadest sense and specifically encompasses 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 a target antigen with a desired specificity and affinity).

[0029] In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., an immunoglobulin) with 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 a 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 contains a variable region (referred to herein as V) at the N-terminus. H The heavy chain constant region is divided into three domains, C H1 , C H2 and C H3 Each light chain consists of a variable region (referred to herein as V I The light chain constant region consists of one domain, C L It consists of. V L is V H Aligned with C L is aligned with the first constant domain (CH1) of the heavy chain. H and V L The pairings of the tetramers together form a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain and thus contain 10 antigen-binding sites, whereas secreted IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units plus the J chain.

[0030] V H and V LBased on structural and sequence analysis, the regions can be further subdivided into regions of hypervariability called hypervariable regions (HVRs). HVRs are interspersed with more conserved regions called framework regions (FWs) (see, e.g., Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). H and V L is composed of three HVRs and four FWs, arranged from amino-terminus to carboxy-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. Similarly, the three HVRs of the light chain are referred to as HVR_L1, HVR_L2, and HVR_L3.

[0031] Table I below provides exemplary CDR definitions according to various algorithms known in the art. TIFF2025509272000001.tif55170 1 Residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest"(1991). 2 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). 3Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. 262:732-745(1996); Abhinandan and Martin, Mol. Immunol.,45:3832-3839(2008). 4 Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., 27:55-77(2003); and Honegger and Plueckthun, J. Mol. Biol., 309:657-670(2001). 5 Residue numbering follows the nomenclature of Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001).

[0032] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2001). nd (ed. Raven Press, NY) (1989).

[0033] L chains from any vertebrate can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, antibodies can be assigned to different classes or isotypes. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, with heavy chains called α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, by the gamma heavy chains Y1-Y4, respectively.

[0034] The terms "antigen-binding fragment," "antigen-binding portion," or "antigen-binding domain" of an antibody, as used interchangeably herein, 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 (i) Fab fragments, V L , V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region, and (iii) a V(ab')2 fragment, a monovalent fragment consisting of the V(ab')2 domain of a single arm of an antibody. L and V H Other exemplary antigen-binding fragments include, but are not limited to, Fv fragments consisting of the F domain. Other exemplary antigen-binding fragments are described elsewhere herein.

[0035] The term "masked antibody" refers to an antibody or antigen-binding fragment thereof that includes a masking peptide that prevents, inhibits, reduces, prevents, inhibits, or competes with the ability of the antigen-binding domain of the antibody to bind to its target. A masked antibody may be generated by attaching a masking peptide to the antigen-binding domain of the antibody. In some embodiments, the masked antibody or antigen-binding fragment thereof exhibits a first binding affinity to the target when in an inactivated state (e.g., inhibited or masked by the masking peptide) and a second binding affinity to the target when in an activated state (e.g., not inhibited or masked by the masking peptide (e.g., the masking peptide has been cleaved from the antibody)), the second binding affinity being greater than the first binding affinity. A masked antibody may be generated by attaching a masking peptide that includes an activatable moiety (e.g., a cleavable site in a linking unit, or "LU") to the antigen-binding domain of the antibody.

[0036] "Masking peptide" refers to a peptide that inhibits an antigen-binding domain from binding to a target antigen, and typically comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linkage unit (LU). The C-terminus of the masking peptide is usually attached to the N-terminus. The VH or VL terminus of the antigen-binding domain. In some embodiments, the masking peptide or a portion thereof interferes with or inhibits the binding of the antigen-binding domain to its target so efficiently that the binding of the antigen-binding domain to its target is extremely low and / or below the limit of detection (e.g., the binding is not detectable by ELISA or flow cytometry assay). The masked antibodies or polypeptides described herein can include one or more linkers, for example, located within the LU, between the MU and the LU, between the LU and the VH or VL, or between the VH and the hinge region of the Fc.

[0037] The LU of the masking peptide may include at least one cleavable site. A cleavage site generally includes a cleavable amino acid sequence, e.g., an amino acid sequence that serves as a substrate for an enzyme, and / or a cysteine-cysteine ​​pair that can form a reducible disulfide bond. Thus, when terms such as "cleavage," "cleavable," "cleaved," and the like are used in connection with a cleavage site, these terms include not only enzymatic cleavage, e.g., by a protease, but also disruption of the disulfide bond between the cysteine-cysteine ​​pair by reduction of the disulfide bond, which may result from exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with or be contained within the MU. A masked antibody or masked polypeptide may include a cleavage site configured to mediate activation of the antibody or polypeptide. For example, if the cleavage site of an activatable antibody is intact (e.g., includes a cysteine-cysteine ​​disulfide bond that is not cleaved and / or reduced by the corresponding enzyme), the masking peptide, or a portion thereof, may interfere with or inhibit binding of the antigen-binding domain to its target. In some embodiments, the LU of the masking peptide does not contain a cleavable site.

[0038] The term "masking efficiency" refers to the efficiency with which a masking peptide inhibits binding of an antigen-binding domain to a target antigen. Masking efficiency can be measured as the difference or ratio of the binding affinity of a masked antibody or masked polypeptide comprising the antigen-binding domain to the binding affinity of an unmasked antibody or unmasked polypeptide comprising the antigen-binding domain (e.g., where the masking peptide has been cleaved from the antibody). For example, masking efficiency can be expressed as the EC50 or K of a masked antibody binding to a target antigen in an inactivated (e.g., inhibited, masked, and / or uncleaved) state. D is expressed as the EC50 or K of the unmasked antibody binding to the target antigen in an activated (e.g., uninhibited, unmasked, and / or cleaved) state. Dor the EC50 or K of the parent antibody (e.g., not linked to a masking peptide) that binds to the target antigen D The EC50 value can be measured by dividing the EC50 value by the ELISA assay or the Jurkat NFAT reporter assay, for example as described in U.S. Patent No. 6,363,950. App. Pub. No. US20210207126A1. D The value can be measured, for example, using surface plasmon resonance.

[0039] The term "epitope" refers to a portion of an antigen to which an antibody (or an antigen-binding fragment thereof) binds. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include a variable number of amino acids in unique spatial conformations. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, 2D nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or any method used in combination with a computational model of the antigen and its complex structure with its bound antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once the desired epitope of an antigen is determined, antibodies against that epitope can be generated, for example, using the techniques described herein. Antibody generation and characterization can also elucidate information about the desired epitope. From this information, it is possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to perform cross-competition studies to find antibodies that competitively bind to each other, i.e., the antibodies compete for binding to the antigen. A high-throughput process for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO03 / 48731.

[0040] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments as passed from parents to offspring via reproductive cells. Germline sequences are distinguished from the nucleotide sequences encoding antibodies in mature B cells that have been altered by recombination and hypermutation events during B cell maturation.

[0041] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a location for the addition of a sugar residue. The amino acids to which carbohydrates, such as oligosaccharides, are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. A particular attachment site is typically indicated 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 standard amino acid except proline. The terms "N-linked" and "O-linked" refer to the chemical groups that serve as the attachment site between a sugar molecule and an amino acid residue. N-linked sugars are added through an amino group and O-linked sugars are added through a hydroxyl group. The term "glycan occupancy" refers to the presence of a sugar moiety linked to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, either none (0-glycan site occupied), one (1-glycan site occupied), or both (2-glycan site occupied) sites can be occupied by a sugar moiety.

[0042] The term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; human cells (e.g., HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells), as well as cells contained within transgenic animals or cultured tissues. This term encompasses not only the particular subject cell, but also the progeny of such cells. Since certain modifications may occur in subsequent generations due to either mutations or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

[0043] A "human antibody" is one that possesses the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, or an amino acid sequence that corresponds to other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0044] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody can contain some or all of the CDRs or HVRs from a non-human animal or synthetic antibody, and the antibody framework and constant regions contain amino acid residues derived from human antibody sequences.

[0045] The term "exemplary antibody" refers to any one of the antibodies described herein. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). L and V H The present invention encompasses antibodies of all five classes that have identical amino acid sequences for the V domain. Furthermore, antibodies of the IgG class can be of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, antibodies of each of the IgG subclasses identified above include antibodies of the V domain and V subclass. L and V H The present disclosure includes antibodies of all four subclasses that have identical amino acid sequences for the heavy chain constant regions. The amino acid sequences of the five classes and four IgG subclasses of human antibodies are known in the art. The amino acid sequences of the full-length heavy and light chains of each of the IgG4 subclasses of exemplary antibodies shown in Table 1b are provided in the present disclosure.

[0046] An "isolated" antibody or binding molecule is one that is isolated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0047] "k a The term "k" refers to the association rate constant of a particular antibody-antigen interaction. d The term "dissociation rate constant" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0048] "K D The term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. This is k d vs. k a The ratio of (i.e., k d / k a ) and expressed as molar concentration (M). D is used as a measure of the affinity of binding of an antibody to a binding partner. D The smaller the K, the stronger 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 to a particular antigen more strongly than an antibody with a micromolar (μM) dissociation constant. D Values ​​can be determined using methods well established in the art. D One way to determine is by using an ELISA. For example, an ELISA-based assay procedure.

[0049] The term "mammal" refers to any animal species of the mammalian class. Examples of mammals include humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; farm animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0050] The terms "prevent" or "preventing" in reference to a particular disease state in a mammal refer to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

[0051] As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are identical between sequences.The amino acid sequence identity of polypeptides can be conventionally determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, 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 using Bestfit or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, allowing for gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence. This aforementioned method of determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants disclosed herein.

[0052] As used herein, the terms "bind", "bind to", "specifically bind to", 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 biological molecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for longer 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 radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. For example, a masked anti-CD137 antibody described herein is said to selectively bind human CD137 if it binds to human CD137 with an EC50 that is less than 10 percent of the EC50 for binding to a different antigen in an in vitro assay.

[0053] The terms "treat", "treating" or "treatment" in reference to a particular disease condition in a mammal refer to causing a desired or beneficial effect in a mammal having the disease condition. A 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 immune cell numbers and / or activity, etc.), or a halt or inhibition of further progression of the disease, condition, or disorder. In the context of cancer treatment in a mammal, a desired or beneficial effect may include inhibition of further growth or spread of cancer cells, killing of cancer cells, inhibition of recurrence of cancer, reduction of pain associated with cancer, or improved survival of the mammal. The effect may be either subjective or objective. For example, if the mammal is a human, the human may note improved energy or vitality, or reduced pain as subjective symptoms of an improvement or response to treatment. Alternatively, the clinician may note a reduction in tumor size or tumor burden based on physical exam, laboratory values, tumor markers, or x-ray findings. Some laboratory signs that a clinician may observe for a response to treatment include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, a clinician may observe a decrease in detectable tumor markers. Alternatively, other tests, such as sonograms, nuclear magnetic resonance studies, and positron emission tests, may be used to assess objective improvement.

[0054] The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be propagated, selected, further manipulated, and / or expressed in a host cell or organism. A vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. One type of vector integrates into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors). Another type of vector is capable of autonomous replication in the host cell into which it is introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Another specific type of vector capable of inducing the expression of an expressible foreign nucleic acid to which they are operably linked is generally called an "expression vector". Expression vectors generally have control sequences that drive the expression of an expressible foreign nucleic acid. Simpler vectors, known as "transcription vectors", are only capable of transcription and not translation: they can replicate in a target cell but not express. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of inducing expression of an expressible nucleic acid to which they are operatively linked are generally referred to as "expression vectors." Other examples of "vectors" can include display vectors (e.g., vectors that direct the expression and display of an encoded polypeptide on the surface of a virus or a cell, such as a bacterial cell, yeast cell, insect cell, and / or mammalian cell).

[0055] As used herein, a "subject," "patient," or "individual" may refer to a human or non-human animal. A "non-human animal" may refer to any animal not classified as a human, such as farm animals, livestock, or zoo animals, sport animals, pet animals (e.g., dogs, horses, cats, cows, etc.), and animals used in research. A research animal may refer to, but is not limited 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.). In some embodiments, the subject, patient, or individual is a human.

[0056] "Effective amount" refers to at least an amount effective at the dosage and for the period of time necessary to achieve one or more desired or indicated effects, including therapeutic or prophylactic results. An effective amount may be provided in one or more administrations. For purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an 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 when administered as a monotherapy or combination therapy). Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved when combined with one or more other agents.

[0057] The methods and techniques of the present disclosure are generally performed according to methods well known in the art and described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. 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's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0058] As used herein, the twenty conventional amino acids and their abbreviations follow standard usage, see Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0059] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0060] Masked anti-CD137 antibodyIn one embodiment, a masking peptide (MP) and human CD137 A masked antibody is provided, comprising an antibody that binds to (hCD137), 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, and the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7, wherein the VH comprises CDR-H1 represented by TGGVGVG (SEQ ID NO: 36), CDR-H2 represented by LIDWADDKYYSPSLKS (SEQ ID NO: 37), and CDR-H3 represented by GGSDTVIGDWFAY (SEQ ID NO: 38), and the VL comprises CDR-L1 represented by RASQSIGSYLA (SEQ ID NO: 39), CDR-L2 represented by DASNLET (SEQ ID NO: 40), and CDR-L3 represented by QQGYYLWT (SEQ ID NO: 41). In some embodiments, a masked antibody is provided comprising a masking 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 masking unit (MU), a linking unit (LU), wherein the VH comprises CDR-H1 set forth in TSGVGVG (SEQ ID NO: 42), CDR-H2 set forth in LIDWDDDKYYSPSLKS (SEQ ID NO: 43), and CDR-H3 set forth in GGSDTVLGDWFAY (SEQ ID NO: 44), and wherein the VL comprises CDR-L1 set forth in RASQSVSPYLA (SEQ ID NO: 45), CDR-L2 set forth in DASSLES (SEQ ID NO: 46), and CDR-L3 set forth in QQGYSLWT (SEQ ID NO: 47).In some embodiments, a masked antibody is provided comprising a masking peptide (MP) and an antibody that binds 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 masking unit (MU), a linking unit (LU), wherein the VH comprises CDR-H1 set forth in SGHYWA (SEQ ID NO: 48), CDR-H2 set forth in SISGYGSTTYYADSVKG (SEQ ID NO: 49), and CDR-H3 set forth in GGSDAVLGDWFAY (SEQ ID NO: 50), wherein the VL comprises CDR-L1 set forth in RASQGIGSFLA (SEQ ID NO: 51), CDR-L2 set forth in DASNLET (SEQ ID NO: 40), and CDR-L3 set forth in QQGYYLWT (SEQ ID NO: 41). SEQ ID NOs: 1-7 are shown in Table 2 below: TIFF2025509272000002.tif62170

[0061] In some embodiments, the MP further comprises an N-terminal unit. In some embodiments, the N-terminal unit is about 1-10 amino acids in length. In some embodiments, the N-terminal unit comprises E (glutamic acid) or EVGSY (SEQ ID NO: 77). In some embodiments, the LU comprises at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the LU further comprises a second cleavage site (CS2). In some embodiments, the first cleavage site and / or the second cleavage site are protease cleavage sites. In some embodiments, the first cleavage site and the second cleavage site 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-exist with the target of an activatable antibody that contains a cleavage site) may be used, including, for example, 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, ADAMTS1, ADAMTS4, and / or ADAMTS5), caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and / or caspase-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 ​​proteinases (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., meprin, neprilysin, PSMA, and / or BMP-1), serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteinases). These include protease cleavage sites recognized and / or cleaved by protease (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, tPA, tryptase, type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4), and the like. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from 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, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from 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).

[0062] In some embodiments, LU further comprises a first linker (L1). In some embodiments, the first linker (L1) is C-terminal to the first cleavage site (CS1) (e.g., the first protease cleavage site). In some embodiments, LU comprises, from the N-terminus to the C-terminus, the structure (CS1)-L1. In some embodiments, LU further comprises a second linker (L2). In some embodiments, L2 is C-terminal to the second cleavage site. In some embodiments, LU comprises, from the N-terminus to the C-terminus, the structure (CS1)-L1-(CS2)-L2. In some embodiments, L1 and L2 are any suitable linker (e.g., flexible linker) known in the art, including, but not limited to, for example, glycine polymer (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), where 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 GGGS (SEQ ID NO: 108), GGGT (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); glycine-alanine polymers; alanine-serine polymers, etc. The linker sequence may be any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20 amino acid glycine polymer or glycine-serine polymer), from about 1 amino acid to about 15 amino acids, from about 3 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from 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.

[0063] In some embodiments, the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 10 to 16. SEQ ID NOs: 8 and 10 to 16 are shown in Table 3 below. TIFF2025509272000003.tif69170

[0064] In some embodiments, the masking peptide (MP) comprises, from the N-terminus to the C-terminus, the structure (MU)-(LU), where LU comprises the structure (CS1)-L1_ or (CS1)-L1-(CS2)-L2. In some embodiments, the masking peptide of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-65.

[0065] In some embodiments, the masking peptide (MP) comprises an MU set forth in any one of SEQ ID NOs: 1-7 and an LU set forth in any one of SEQ ID NOs: 8-16. In some embodiments, the MP comprises a sequence set forth in any one of SEQ ID NOs: 17-35. SEQ ID NOs: 17-35 are shown in Table 4 below. TIFF2025509272000004.tif160170 * The N-terminal unit sequence is in plain text. Masking sequences are bold and underlined. Linkage units are shown in bold.

[0066] In some embodiments, the masked antibody (also referred to herein as a "masked anti-CD137 antibody") comprises a VH set forth in SEQ ID NO: 52 and a VL set forth in SEQ ID NO: 53. In some embodiments, the masked antibody (also referred to herein as a "masked anti-CD137 antibody") comprises a VH set forth in SEQ ID NO: 54 and a VL set forth in SEQ ID NO: 55. In some embodiments, the masked antibody (also referred to herein as a "masked anti-CD137 antibody") comprises a VH set forth in SEQ ID NO: 56 and a VL set forth in SEQ ID NO: 57. In some embodiments, the masked anti-CD137 antibody comprises a VH set forth in SEQ ID NO: 52 and a VL set forth in any one of SEQ ID NOs: 58-76. SEQ ID NOs: 52-76 are shown in Tables 5A and 5B below. TIFF2025509272000005.tif102170TIFF2025509272000006.tif250170TIFF2025509272000007.tif166170*N-terminal unit is plain text, Masking units are underlined and bold text, Linkage units are in bold text, VL sequences are in italic text.

[0067] In some embodiments, the masked anti-CD137 antibody comprises an MP comprising SEQ ID NO: 34, a VH comprising CDR-H1 represented by TGGVGVG (SEQ ID NO: 36), CDR-H2 represented by LIDWADDKYYSPSLKS (SEQ ID NO: 37), and CDR-H3 represented by GGSDTVIGDWFAY (SEQ ID NO: 38), and a VL comprising CDR-L1 represented by RASQSIGSYLA (SEQ ID NO: 39), CDR-L2 represented by DASNLET (SEQ ID NO: 40), and CDR-L3 represented by 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.

[0068] In some embodiments, the masked anti-CD137 antibody comprises a full-length antibody light chain, e.g., a kappa light chain or a lambda light chain. Additionally or alternatively, in some embodiments, the anti-CD137 antibody comprises a full-length antibody heavy chain. The antibody heavy chain may be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is of the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein can be converted from one class or subclass to another class or subclass by methods known in the art. In some embodiments, the masked anti-CD137 antibody is or comprises a full-length antibody comprising an Fc region, e.g., 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, the variant Fc region comprises one or more amino acid substitutions, insertions, or deletions compared to the wild-type human Fc region from which the variant is derived. In some embodiments, the masked anti-CD137 antibody comprises a variant of a human IgG1 Fc region. In some embodiments, the IgG1 Fc variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant for FcγRIIb. In some embodiments, the variant of the human IgG1 Fc region 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, with amino acid numbering according to the EU index. (See, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85). For the above substitutions, see Chu et al. (2008) Mol Immunol.45 (15): 3926-33. Additionally or alternatively, in some embodiments, the variants of the human IgG1 Fc region are 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; G237D, P238 and E233D, G237D, P238D, H268D, P238D, P271G, and A330R, with amino acid numbering according to the EU index. For the foregoing substitutions, see Mimoto et al. (2013) Protein Engineering Selection. 26 (10): 589-98. Additionally or alternatively, in some embodiments, the variant of the human IgG1 Fc region comprises a S2657A substitution (Buschor et al. (2014) Int Arch Allergy Immunol. 163 (3): 206-14), with amino acid numbering according to the EU index. Additionally or alternatively, in some embodiments, the variant of the human IgG1 Fc region comprises a T437R and / or a K248E substitution (Zhang et al. (2017) MAbs. 9 (7): 1129-1142), where amino acid numbering is according to the EU index. In some embodiments, the masked anti-CD137 antibody comprises a variant of a human IgG4 Fc region. In some embodiments, the IgG4 Fc variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant for FcγRIIb. In some embodiments, the variant of the human IgG4 Fc region 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, where amino acid numbering is according to the EU index.Additionally or alternatively, in some embodiments, the variants of the human IgG4 Fc region are 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, P27 1G, and A330R; E233D, H268D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; and E233D, G237D, P238D, H268D, P271G, and A330R; and E233D, G237D, P238D, H268D, P271G, and A330R and K248E, where the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the variant human IgG4 Fc region comprises a S2657A substitution, where the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the variant human IgG1 Fc region comprises a T437R and / or K248E substitution, where the amino acid numbering is according to the EU index.

[0069] In some embodiments, the masked anti-CD137 antibody comprises a masking peptide of SEQ ID NO: 34, a VH domain set forth in SEQ ID NO: 52, and a VL domain set forth in SEQ ID NO: 53. In some embodiments, the masked anti-CD137 antibody further comprises a human IgG1 domain or a variant thereof comprising one or more substitution mutations. In some embodiments, the IgG1 variants are 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, H2 and E233D, G237D, P238D, H268D, P238D, P271G, and A330R; S2657A; T437R; K248E; and T437R and K248E, where amino acid numbering is according to the EU index. In some embodiments, the masked anti-CD137 antibody further comprises a human IgG4 domain or a variant thereof comprising one or more substitution mutations.In some embodiments, the IgG4 variants are 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, H2 and E233D, G237D, 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, where amino acid numbering is according to the EU index.

[0070] In some embodiments, the masked anti-CD137 antibody comprises a heavy chain constant region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111-118. See Table 6 below. TIFF2025509272000008.tif219170TIFF2025509272000009.tif126170

[0071] 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.

[0072] 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 shown below in Tables 7A and 7B. JPEG2025509272000010.jpg247170JPEG2025509272000011.jpg231170 For LC, the VL sequence is in italic text, CL is in double underlined text For HC, the VH sequence is in italic text; CH is in double underlined text JPEG2025509272000012.jpg222170JPEG2025509272000013.jpg240170JPEG2025509272000014.jpg240170JPEG2025509272000015 .jpg240170JPEG2025509272000016.jpg240170JPEG2025509272000017.jpg250170JPEG2025509272000018.jpg232170JPEG202550 9272000019.jpg250170JPEG2025509272000020.jpg232170JPEG2025509272000021.jpg228170JPEG2025509272000022.jpg250170JPEG2025509272000023.jpg227170JPEG2025509272000024.jpg245170JPEG2025509272000025.jpg82170· For LC, the N-terminal unit is plaintext; Masking units are underlined bold text, Linkage units are in bold text, VL sequences are in italic text, CL is in double underlined text For HC, the VH sequence is in italic text; CH is in double underlined text

[0073] In some embodiments, the term "masked anti-CD137 antibody" refers to an antibody fragment, e.g., a masked antigen-binding fragment of a masked anti-CD137 antibody. In some embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv (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) VHH, minibody, or diabody.

[0074] In some embodiments, the masked anti-CD137 antibodies described herein cross-react with CD137 of different species, thus allowing the masked anti-CD137 antibodies to be used in both preclinical studies and clinical trials. In some embodiments, the masked anti-CD137 antibodies described herein bind to two or more of human CD137, cynomolgus monkey CD137, mouse CD137, and / or rat CD137 after activation (i.e., after activation by cleavage of the masked antibody, e.g., protease cleavage). In some embodiments, the masked anti-CD137 antibodies bind to human CD137, cynomolgus monkey CD137, mouse CD137, and rat CD137 after activation (i.e., after activation by cleavage of the masked antibody, e.g., protease cleavage).

[0075] In some embodiments, the masked anti-CD137 antibodies described herein are context-dependent (e.g., are only activated (capable of binding to a target) in certain contexts (such as a protease-rich tumor microenvironment). In some embodiments, the masked anti-CD137 antibodies described herein have improved safety (e.g., have reduced toxicity, do not significantly change the weight of many organs, do not alter liver histopathology, hematology, and / or blood biochemistry, etc.) over more traditional unmasked antibodies. In some embodiments, the masked anti-CD137 antibodies described herein exhibit similar pharmacokinetic properties (e.g., have similar in vivo half-lives) as traditional unmasked anti-CD137 antibodies. In some embodiments, the masked anti-CD137 antibodies described herein exhibit improved pharmacokinetic properties (e.g., have longer in vivo half-lives) compared to more traditional unmasked anti-CD137 antibodies.

[0076] 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 hCD137. In some embodiments, the masking unit (MU) of the masked anti-CD137 antibodies described herein binds to the ABD of hCD137 and reduces or inhibits binding of the masked anti-CD137 antibody to hCD137 compared to binding of a corresponding anti-CD137 antibody lacking the MU to hCD137 and / or compared to binding of the ABD to hCD137. In some embodiments, the masking units (MU) measures 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 1,000, at least about 1,100, at least about 1,200, at least about 1,300, at least about 1,400, at least about 1,500, etc., including any ranges between these values) prior to removing MU from the masked anti-CD137 antibody. In some embodiments, masking efficiency is measured as the difference between the binding affinity of a masked anti-CD137 antibody comprising a masking unit (MU) to hCD137 (i.e., before activation of the masked antibody) and the binding affinity of an anti-CD137 antibody lacking MU to hCD137. In some embodiments, masking efficiency is measured as the difference between the affinity of a masked anti-CD137 antibody comprising MU to hCD137 (i.e., before activation of the masked antibody by cleavage, e.g., protease cleavage) and the affinity of an unmasked anti-CD137 antibody to hCD137 (i.e., after activation of the masked antibody by cleavage, e.g., protease cleavage). In some embodiments, masking efficiency is measured as the EC for binding of a masked anti-CD137 antibody comprising MU 50(i.e., before activation) were compared with the EC 50 In some embodiments, the EC 50 is measured by ELISA. In some embodiments, the masking unit (MU) of the masked anti-CD137 antibody binds to the ABD and prevents the masked anti-CD137 polypeptide from binding to hCD137.

[0077] In some embodiments, the affinity of a masked anti-CD137 antibody of the 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). When the masking unit is removed from the antibody (e.g., after activation by treatment with one or more proteases that cleave within the linking unit), the inhibitory effect on the anti-CD137 antibody is improved by 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 range between the aforementioned values)) compared to a corresponding anti-CD137 antibody without the masking peptide or masking unit. In some embodiments, the EC 50is 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 aforementioned values) after activation by treatment with one or more proteases that cleave within a binding unit, e.g., as measured by ELISA or FACS assay.

[0078] In some embodiments, when a masking unit is attached to the ABD of a masked anti-CD137 antibody described herein, the K D indicates that if the masking unit of a masked anti-CD137 antibody is removed from the ABD (e.g., cleaved within the target by protease treatment), the K D about 2-fold (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 aforementioned values) greater than the K of the antibody against its target. D is the K of the corresponding anti-CD137 antibody lacking the masking peptide or masking unit. Dabout twice (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 aforementioned values).

[0079] In some embodiments, the masking unit sterically hinders the binding of the masked binding polypeptide to its target and / or allosterically hinders the binding of the masked binding polypeptide to its target.

[0080] In some embodiments, the dissociation constant of the masking unit of a masked anti-CD137 antibody described herein with respect to the ABD is greater than the dissociation constant of the masked anti-CD137 antibody with respect to hCD137 (when the masked anti-CD137 antibody is in an active form, such as after protease treatment). In some embodiments, the dissociation constant of the masking unit with the ABD of a masked anti-CD137 antibody described herein is greater than about twice (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 aforementioned values) the dissociation constant of the masked anti-CD137 antibody with hCD137 (when the masked anti-CD137 antibody is in an active form, such as after protease treatment). In some embodiments, the dissociation constant of the masking unit with respect to the ABD of the masked anti-CD137 antibody described herein is approximately equal to the dissociation constant of the masked anti-CD137 antibody with respect to hCD137 (when the masked anti-CD137 antibody is in an activated form, such as after protease treatment). In some embodiments, the masking unit (MU) binds to the ABD of the masked anti-CD137 antibody described herein and prevents the antibody from binding to hCD137 only if the masked anti-CD137 antibody is not activated (e.g., by treatment with one or more proteases that cleave within the linking unit). In some embodiments, activation induces cleavage of the polypeptide within the cleavage site. In some embodiments, activation induces a conformational change in the polypeptide (e.g., displacement of the masking unit (MU)) such that the masking peptide no longer prevents the polypeptide from binding to the target.

[0081] The masked antibodies described herein may be further modified. In some embodiments, the masked antibodies are linked to additional molecular entities. Examples of additional molecular entities include pharmaceuticals, peptides or proteins, detection agents or labels, antibodies, etc.

[0082] In some embodiments, the activatable binding polypeptides of the present disclosure are linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, corticine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclosporine, 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, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine. 131 ,indium 111 ,yttrium 90 , and lutetium 177Methods of linking a polypeptide to a pharmaceutical agent are known in the art, including, but not limited to, using a variety of linker technologies. Exemplary linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For further discussion of linkers and methods for linking 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.

[0083] Nucleic acids, vectors, host cells, and recombinant methods for producing targeted anti-CD137 antibodies Another aspect of the present disclosure provides one or more isolated nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence of the masked anti-CD137 antibody described herein. The amino acid sequence encoded by the nucleotide sequence can be any part of the masked anti-CD137 antibody, such as CDR, a sequence comprising one, two or three CDRs, the variable region of the heavy chain, the variable region of the light chain, or the full-length heavy chain or full-length light chain. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not include intron sequences. Usually, the nucleic acid is a cDNA molecule.

[0084] In some embodiments, the present disclosure provides isolated nucleic acid molecules comprising or consisting of a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region and / or the light chain variable region of, e.g., a masked anti-CD137 antibody described herein, or the full-length heavy chain and / or full-length light chain of, e.g., a masked anti-CD137 antibody described herein.

[0085] The nucleic acids of the invention can be obtained using any suitable molecular biology technique, such as PCR amplification or cDNA cloning techniques. In the case of masked anti-CD137 antibodies, the nucleic acids encoding the antibodies obtained by library screening can be recovered from the library.

[0086] V H The isolated DNA encoding the region is H A full-length heavy chain gene can be obtained by operably linking the DNA encoding the heavy chain constant region (CH1, CH2, and CH3) to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG4 or IgG2 constant region with no ADCC effect. The IgG4 constant region sequence can be any of the various alleles or allotypes known to occur among different individuals. These allotypes represent naturally occurring amino acid substitutions in the IgG4 constant region. In the case of a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0087] V L The isolated DNA encoding the region is L The DNA encoding the light chain constant region, CL, can be converted to a full-length light chain gene by operably linking the DNA encoding the light chain constant region, CL, to another DNA molecule encoding the light chain constant region. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing 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 masked anti-CD137 antibody comprises a light chain constant region as set forth in SEQ ID NO:111, as shown below: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 111)

[0088] To generate the scFv gene, H and V L The DNA fragment encoding V H and V L The array is V L and V HThe region is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:128), so that it can be expressed as a contiguous single-chain protein joined by a flexible linker (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)).

[0089] The present invention further provides vectors comprising one or more of the nucleic acid molecules provided by the present invention. In some embodiments, the vector is an expression vector useful for expressing a masked anti-CD137 antibody or a masked antigen-binding fragment of such an antibody. In some embodiments, vectors are provided, where a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein and a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises polynucleotides encoding a heavy chain variable region as described herein and a light chain variable region as described herein.

[0090] To express the binding molecules of the present disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector such that the DNA molecule is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is linked to a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or homologous recombination-based DNA ligation). The light and heavy chain variable regions of the antibodies described herein are inserted into an expression vector already encoding the heavy and light chain constant regions of the desired isotype and subclass, and the V H The segment is C in the vector H operably linked to the segment(s), L The segment is C in the vector L The recombinant expression vector can be used to generate full-length antibody genes of any antibody isotype and subclass by operably linking the antibody chain to a segment. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0091] In addition to the antibody chain genes, the expression vectors of the disclosure typically carry regulatory sequences that control the expression of the antibody chain genes in a 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 genes. Such regulatory sequences are 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, can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP) and polyoma). Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the β-globin promoter. Additionally, regulatory elements consist 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).

[0092] In addition to the antibody chain genes and regulatory sequences, expression vectors can carry additional sequences, such as enhancer elements, transcription termination sequences, sequences that regulate replication of the vector in a host cell (such as origins of replication), and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0093] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by any suitable technique. The various forms of the term "transfection" are intended to encompass 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. The masked anti-CD137 antibodies described herein can be expressed in either prokaryotic or eukaryotic host cells, although expression of the antibodies in eukaryotic cells, such as mammalian host cells, is most common.

[0094] The present invention further provides a host cell comprising the nucleic acid molecule or vector provided by the present invention. 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, or a prokaryotic cell such as a bacterial cell. The introduction of the recombinant nucleic acid construct into the host cell can be achieved by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

[0095] Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0096] Mammalian host cells for expressing the binding molecules of the disclosure include, for example, Chinese hamster ovary (CHO) cells (including, for example, dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), used with DHFR, as described in Kaufman and Sharp, J. Mol. Biol. 159:601-621 (1982)), NS0 myeloma cells, COS cells and Sp2 cells. Another expression system, particularly for use with NS0 myeloma or CHO cells, is the GS (glutamine synthetase) gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841.

[0097] The masked anti-CD137 antibodies (or antigen-binding fragments thereof) of the present invention can be produced by any means known in the art. Exemplary techniques for antibody production are described in U.S. Pat. No. 4,816,567, however, these exemplary techniques are provided for illustrative purposes only and are not limiting. Once a nucleic acid or expression vector encoding a masked anti-CD137 antibody is introduced into a host cell, the masked anti-CD137 antibody is produced by culturing the host cell for a period of time sufficient for the antibody to be expressed within the host cell or secreted into the culture medium in which the host cell is grown. Thus, in some embodiments, a method of producing a masked 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 masked anti-CD137 antibody (e.g., those provided above) under conditions suitable for expression of the masked antibody. In some embodiments, the method further comprises recovering the masked anti-CD137 antibody from the host cell (or host cell culture medium). The masked anti-CD137 antibody can be recovered from the culture medium using any suitable protein purification method.

[0098] Pharmaceutical Compositions In another aspect, the present disclosure provides a composition comprising one or more masked anti-CD137 antibodies described herein. In some embodiments, the composition is a pharmaceutical composition comprising a masked anti-CD137 antibody described herein and a pharma- ceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0099] The term "pharmaceutically acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of a polypeptide (e.g., a masked antibody). Carriers can be anti-adherents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption retardants, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), glucose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0100] The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. Particular forms of the composition suitable for delivering the masked anti-CD137 antibody are sterile liquids, such as solutions, suspensions, or dispersions for injection or infusion. Sterile solutions can be prepared by incorporating the required amount of the masked anti-CD137 antibody in a suitable carrier, followed by sterile microfiltration. Dispersions are prepared by incorporating the masked anti-CD137 antibody into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, the preparation method includes vacuum drying and freeze-drying (lyophilization) to obtain a powder containing the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. Various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0101] The relative amount of masked anti-CD137 antibody included in the composition will vary depending on many factors, such as the particular polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of masked anti-CD137 antibody in a single dosage form will generally be that amount that provides a therapeutic effect, but may be a lesser amount. Generally, this amount will range from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent, based on the total weight of the dosage form.

[0102] In addition to the masked anti-CD137 antibody, one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described in WO 2019 / 037711, the contents of which are incorporated herein by reference in their entirety. The suitable amount of additional therapeutic agent to be 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 drug and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form is generally the amount of drug that produces a therapeutic effect, but may be a smaller amount.

[0103] Any of the masked anti-CD137 antibodies and / or compositions (e.g., pharmaceutical compositions) described herein can be used in the preparation of a medicament (e.g., a medicament for use in treating cancer or delaying the progression of cancer in a subject in need thereof).

[0104] Treatment method The masked anti-CD137 antibodies and pharmaceutical compositions described herein are useful for therapeutic purposes, such as treating cancer or improving the effectiveness of other cancer treatments. Thus, in other aspects, the disclosure provides a method of using a masked anti-CD137 antibody or pharmaceutical composition. In one aspect, the disclosure provides a method of treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of a masked anti-CD137 antibody. In some embodiments, the cancer is a solid tumor cancer (e.g., breast cancer, liver cancer, colon cancer, colon cancer, etc.).

[0105] When carrying out the method of treatment, the masked anti-CD137 antibody described herein may be administered alone, i.e., as a monotherapy, or in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the present disclosure provides combination therapy comprising a binding molecule in combination with one or more additional therapeutic agents or therapies for separate, sequential, or simultaneous administration. In some embodiments, the term "additional therapy" refers to a therapy that does not use a masked anti-CD137 antibody as a therapeutic agent. In some embodiments, the term "additional therapeutic agent" refers to any therapeutic agent other than the masked anti-CD137 antibody described herein. In some embodiments, the present disclosure provides a method of treating cancer in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of a masked anti-CD137 antibody described herein and an effective amount of an anti-PD-1 antibody (e.g., an anti-human PD-1 antibody). In some embodiments, the present disclosure provides a method of treating cancer in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of a masked anti-CD137 antibody described herein and an effective amount of an anti-CTLA4 antibody (e.g., an anti-human CTLA4 antibody). In some embodiments, the anti-CTLA4 antibody is a masked anti-CTLA4 antibody. In some embodiments, the disclosure provides a method of treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of a masked anti-CD137 antibody 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, the bispecific antibody that binds HER2 and CD3 is a masked bispecific antibody that binds HER2 and CD3. In some embodiments, the disclosure provides a method of treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of a masked anti-CD137 antibody described herein and an effective amount of a bispecific T cell engager (TCE) that targets 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, 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 domain fusion, the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, the first and second polypeptide chains associate to form a HER2 binding arm, the third polypeptide chain binds to CD3, and 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 masked TCE (e.g., the portion of the TCE that binds to CD3 and / or the portion of the TCE that binds to HER2 is masked). 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, colon cancer, or colon cancer).

[0106] 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 set forth in SEQ ID NO: 52, and a VL domain set forth in SEQ ID NO: 53. In some embodiments, the masked anti-CD137 antibody further comprises a human IgG1 domain or a variant thereof comprising one or more substitution mutations. In some embodiments, the IgG1 variants are 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 P238; E233D, P238D, and A330R; E233D, P231G, P238D, and A330R; G237D, H26 and E233D, G237D, 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, where amino acid numbering is according to the EU index. In some embodiments, the masked anti-CD137 antibody further comprises a human IgG4 domain or variant thereof comprising one or more substitution mutations.In some embodiments, the IgG4 variants are 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, H2 and E233D, G237D, 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, where amino acid numbering is according to the EU index.

[0107] Kits and Products In another aspect, a kit is provided that includes one or more masked anti-CD137 antibodies described herein. In some embodiments, the kit further includes a package insert that includes instructions for use of the masked anti-CD137 antibody. In some embodiments, the article of manufacture or kit includes a container that includes one or more of the masked anti-CD137 antibodies or compositions described herein. In certain embodiments, the article of manufacture or kit includes a container that includes a nucleic acid encoding one (or more) of the masked anti-CD137 antibodies described herein. In some embodiments, the kit includes cells of a cell line that produces a masked anti-CD137 antibody described herein. In some embodiments, the kit includes CD137 (e.g., human CD137, cynomolgus monkey CD137, mouse CD137, rat CD137, or a fragment of any of the foregoing) or CD137 +One or more positive controls for the cells are included. In some embodiments, the kit includes a negative control, e.g., a surface or solution that is substantially free of CD137, or cells that do not express CD137.

[0108] In certain embodiments, the article of manufacture or kit includes 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 masked anti-CD137 antibody is used to treat solid cancer. For example, a tumor (e.g., breast cancer, liver cancer, colon cancer, colon cancer, etc.) is treated in a subject (e.g., a human subject) in need thereof according to the methods provided herein. Suitable containers include, for example, bottles, vials, syringes, intravenous fluid bags, test tubes, and the like. The containers are formed from a variety of materials, including glass and plastic. The containers contain a masked anti-CD137 antibody (or a composition comprising such a masked antibody) that is effective, alone or in combination with other compositions, in treating, delaying the progression of, and / or preventing cancer in a subject (e.g., a human subject). The container may be provided with a sterile access port (e.g., the container may be a vial or an intravenous fluid bag with a stopper that can be pierced by an intravenous needle). In some embodiments, the label or package insert indicates that the composition is used for treating breast cancer, liver cancer, colon cancer, or colon cancer in a subject (eg, a human subject).

[0109] Additionally, the article of manufacture or kit may include (a) a first container containing a composition comprising a masked anti-CD137 antibody (or immunologically active fragment thereof) described herein, and (b) a second container containing a composition, the composition including an additional cytotoxic or other 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 the article of manufacture includes a label or package insert indicating that the masked anti-CD137 antibody and anti-PD-L1 are used to treat a solid tumor (e.g., breast cancer, liver cancer, colon 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 an anti-CTLA4 antibody (e.g., an anti-human CTLA4 antibody, such as a masked anti-CTLA4 antibody), and the article of manufacture includes a label or package insert indicating that the masked anti-CD137 antibody and the anti-CTLA4 antibody (e.g., the masked anti-CTLA4 antibody) are used to treat a solid tumor (e.g., breast cancer, liver 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 masked bispecific antibody that binds HER2 and CD3, and the article of manufacture includes a label or package insert indicating that the masked anti-CD137 antibody and the bispecific antibody that binds HER2 and CD3 (e.g., a masked bispecific antibody that binds HER2 and CD3) are used to treat a solid tumor (e.g., breast cancer, liver cancer, colon cancer, etc.) in a subject (e.g., a human). For example, it is administered to a subject (patient) in need thereof according to the methods provided herein.In some embodiments, the second container contains a composition comprising a bispecific T cell engager (TCE) (e.g., a masked bispecific T cell engager that binds 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, a first polypeptide chain comprises an antibody heavy chain, a second polypeptide chain comprises an antibody light chain, and a third polypeptide chain comprises an scFv-Fc domain fusion, the C-terminus of the ScFv is fused to the N-terminus of the Fc domain, the first and second polypeptide chains associate to form a HER-2 binding arm, the third polypeptide chain binds to CD3, and 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 masked TCE (e.g., the portion of the TCE that binds CD3 and / or the portion of the TCE that binds HER2 is masked). 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 includes a label or package insert indicating that the masked anti-CD137 antibody and 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) are used to treat a solid tumor (e.g., breast cancer, liver cancer, colon cancer, colon cancer, etc.) in a subject (e.g., a human subject) in need thereof, e.g., according to the methods provided herein.

[0110] In addition, the article of manufacture may further comprise additional containers containing pharma- ceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution, etc. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0111] The above description is deemed sufficient to enable those skilled in the art to practice the present disclosure. The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, in addition to the modifications shown and described herein, various modifications of the present disclosure will become apparent to those skilled in the art from the above description and will fall within the scope of the appended claims.

[0112] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0113] The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise noted, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric.

[0114] Example 1: Construction and validation of a masked antibody targeting CD137

[0115] FACS-based screening of masking peptides against CD137 antibodies A total of 1x10 IgG from the constrained peptide library (CPL) library described in US 2019 / 00241886, the contents of which are incorporated herein by reference in their entirety. 8Yeast cells were used to screen masking peptides against the parent anti-CD137 antibody TY21242, which contains the heavy chain variable domain (VH) set forth in SEQ ID NO: 52 and the light chain variable domain (VL) sequences set forth in SEQ ID NO: 53 as shown below (CDR sequences are shown in bold). EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSS (SEQ ID NO:52) DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKR (SEQ ID NO:53)

[0116] In this example, a yeast library was constructed in which the masking units of CPLs were directly fused to the N-terminus of the light chain of the parent antigen-binding domain, respectively, and the fusion proteins were displayed on the yeast cell surface. To identify masking peptide sequences that could effectively mask the parent anti-CD137 antibody, the yeast library was subjected to several rounds of FACS-based screening. First, yeast clones with low binding to human CD137 were enriched, and then the enriched yeast clones were treated with protease to remove the masking units and select clones with high binding to the antigen.

[0117] Briefly, for each round of sorting with the MOFLO XDP® High-Speed ​​Cell Sorter, yeast cells induced in galactose medium were harvested, washed once with PBSA buffer (i.e., 1% BSA in PBS), and then incubated with different concentrations of biotinylated CD137 for 1 h at room temperature. Yeast cells were then washed twice with PBSA buffer and incubated with phycoerythrin (PE)-conjugated streptavidin (1:500 dilution) (eBioscience #2-4317-87) for 30 min at 4 °C. After two further washes with PBSA buffer, yeast cells were adjusted to 2–3 OD / mL and sorted. 1 nM biotinylated CD137-Fc was used in all five sorts, enriching for weak binders in each round. Yeast cells showing weak binding in round 5 were grown in glucose and then induced in galactose medium and treated with TEV protease (6 U / OD cells) (in-house) for 2 h at 30 °C. Yeast cells that showed strong binding after protease treatment were purified. It was clear that TEV cleavage dramatically increased the number of cells that strongly bound the antigen, suggesting that the screening strategy was effective. Single clones obtained from the fifth round of sorting were plated on selective medium and cultured individually for further confirmation of cleavage-mediated activated antigen binding. In the Fab format, the selected masked anti-CD137 antibody clones showed little binding to the antigen (i.e., hCD137) in the presence of the masking peptide. However, when yeast cells were treated with TEV protease to remove the masking peptide, binding to the antigen increased dramatically.

[0118] After five rounds of selection, plasmids were extracted from these clones and the masking unit sequences were confirmed by DNA sequencing. To determine the amino acid sequences of the masking peptides, shuttle plasmids were extracted from selected yeast clones (Generay #GK2002-200) and transformed into competent E. coli cells. Plasmids were prepared and the regions encoding the masking peptides were sequenced and aligned. The sequences of the masking peptides could be divided into several groups, showing clear enrichment through the rounds of sorting. The masking unit sequences and linkage unit sequences of several antibodies are shown in Table A1. The complete sequences of the masking peptides are shown in Table A2. The masking peptides, from the N-terminus to the C-terminus, comprise an N-terminal unit (i.e., amino acid sequence EVGSY (SEQ ID NO: 77)), a masking unit, and a linkage unit. The VH and VL sequences of the antibodies listed in Tables A1 and A2 are shown in Tables 5A and 5B. TIFF2025509272000026.tif62170TIFF2025509272000027.tif62170*N-terminal unit sequence in normal characters, Masking sequences are in bold and underlined text. Linkage unit sequences are shown in bold.

[0119] IgG conversion and expression The sequences of the masking and linkage units of additional masked anti-CD137 antibodies derived from TY22586, TY22594, TY22595, and TY22599 are shown in Table B1 below. The complete sequences of the masking peptides are shown in Table B2. From N-terminus to C-terminus, the masking peptides include an N-terminal unit (i.e., amino acid sequence EVGSY (SEQ ID NO: 77)), a masking unit, and a linking unit. The VH and VL sequences of the antibodies in Tables B1 and B2 are shown in Tables 5A and 5B. The antibodies in Tables B1 and B2 are converted to IgG1 or IgG4 masked antibodies, and each masked antibody contains one or two cleavage sites (uPA and / or matrix metalloproteases (MMPs), such as MMP9). To enhance binding to FcγRIIb of the masked antibodies in Tables B1 and B2, 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 no. K830001), and the masking peptide and the truncated peptide were fused to the N-terminus of the light chain in the same manner as they are displayed on the yeast surface of CPL. TIFF2025509272000028.tif137170TIFF2025509272000029.tif145170*N-terminal unit sequence in normal characters, Masking sequences are in bold and underlined text. Linkage units are shown in bold.

[0120] Plasmid pairs encoding the VH and VL of each masked anti-CD137 antibody (i.e., the N-terminus of each VL was fused to a peptide containing the masking sequence and linking unit) were transiently transfected into HEK293F cells. After 6 days, the supernatants were harvested, clarified by centrifugation and filtration, and the antibodies were purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The masked anti-CD137 antibodies were eluted, neutralized, and buffer-exchanged into 20 mM histidine, pH 5.5 buffer. Protein concentrations were measured by UV spectrophotometry, and antibody purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions. The expression levels of the masked anti-CD137 antibodies in HEK293 cells were comparable or lower than those of the parental antibodies, and the purification yields after protein A resin were also similar to those of the parental antibodies, suggesting that the presence of the masking and cleavage peptides does not have a significant adverse effect on antibody expression in mammalian cells.

[0121] Measurement of masking efficiency The affinity of the masked anti-CD137 antibody for human CD137 displayed on the surface of yeast cells was also evaluated. Briefly, yeast cells were transformed with a plasmid expressing full-length human CD137, followed by a C-terminal 3×Myc tag used to identify transformed cells. The transformed cells were transferred to a 1.5 mL tube, washed once with 1% PBSA buffer, centrifuged, and cultured at a density of 0.2 OD in 1 mL of 1% PBSA buffer. 600 The antibodies were resuspended at 0.5 μg / mL and dispensed into wells of a 96-well plate. Three-fold serial dilutions of the test antibodies were pipetted into the cell-containing wells of the 96-well plate, incubated on ice for 1 hour (protected from light), washed once with 1% PBSA buffer, and incubated with 0.5 μg / mL PE-conjugated mouse anti-human IgG Fc and 2 μg / mL mouse anti-myc-647 for 30 minutes on ice. Cells were washed once before analysis by flow cytometry (Beckman® CytoFlex).

[0122] Experiments for several masked anti-CD137 antibodies were performed multiple times, resulting in two calculated masking efficiencies for each of these masked antibodies. The masking efficiency of each masked antibody was calculated by dividing the KD of the masked antibody's binding to hCD137 by the KD of the parent antibody to hCD137. As shown in Figure 1A-1D and Table C below, each of the masked antibodies had dramatically reduced binding to hCD137 compared to the parent antibody. The calculated masking efficiencies of the masked antibodies ranged from 21 to 1131. Differences in masking efficiency likely arose from variability in measurements and data fitting, and we believe that the masking efficiency of each masked antibody falls within the calculated range. These results indicate that multiple masking peptides identified from CPL maintained masking efficiency when expressed in mammalian cells and when expressed as part of a complete IgG molecule. TIFF2025509272000030.tif255160TIFF2025509272000031.tif8170

[0123] Example 2: Activity of masked anti-CD137 antibodies before and after removal of the masking peptide

[0124] The purified masked antibodies were treated with a protease that recognizes the cleavage sequence within the binding unit. After treatment, the masked antibodies were tested to determine whether removing the masking peptide restored the activity of the antibodies. As an example, 20 µg of TY25366 and TY25368 (0.5 mg / mL) were treated with 1 µg of recombinant human MMP-9 (made in-house) in reaction buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 (w / v), pH 7.5). The reaction was carried out at 37 °C for 24 h. The masking peptide was confirmed to have been removed from the light chain by ELISA and FACS-based assays. As shown in Figure 2A-2B and Table D, after removing the masking peptide, binding of the masked anti-CD137 antibodies to hCD137 was indistinguishable from the parent antibodies (i.e., TY21242 and TY23310). TIFF2025509272000032.tif128170

[0125] Example 3: Developability Profile of Masked Anti-CD137 Antibodies

[0126] For manufacturing purposes, it is important that the masked anti-CD137 antibody has a good developability profile. Different assays were performed with purified masked antibodies expressed in mammalian cells, as detailed in this example. Masked antibodies were adjusted to 1 mg / mL in 20 mM histidine, pH 5.5, and antibody quality analysis was performed using analytical size-exclusion chromatography (SEC) using a Thermo U3000 equipped with a Thermo DAD detector and an XBridge BEH SEC column (7.8 mm × 300 mm) (Waters). For each assay, 40 μg of antibody was injected and fractions were performed at a flow rate of 0.7 mL / min in buffer (50 mM phosphate in 300 mM sodium chloride at pH 6.8). Five accelerated stress tests were performed, as shown in Figure 3A-3E. Briefly, TY25366 and TY25368 were subjected to (1) 0, 3, and 6 freeze-thaw cycles, (2) storage at 40°C for 0, 7, 14, 21, and 28 days, (3) storage at room temperature for 0 and 2 hours in acidic buffer (sodium acetate solution, pH 3.6), (4) storage at 40°C for 0 and 24 hours in 50 mM histidine, 300 mM NaCl, pH 7.0 buffer, and (5) storage at room temperature for 6 hours in saline followed by storage at 4°C for 24 hours. The SEC profiles of TY25366 and TY25368 after storage at accelerated storage conditions were similar to those of TY25366 and TY25368 at T=0 (i.e., before storage under the aforementioned conditions). Since the masked antibodies have not yet undergone an extensive buffer optimization process, it is possible that optimized buffers and excipients could further improve the stability of the masked antibodies. Taken together these data indicate that the masked anti-CD137 antibody remains stable under a range of stress conditions and therefore has a favorable developable profile.

[0127] Example 4: Binding of anti-CD137 antibodies TY24118 and TY24122 to FcγR

[0128] The binding of anti-CD137 antibodies TY24118 and TY24122 (i.e., parent antibodies of masked antibodies TY25368 and TY25366, respectively) to Fcγ receptors was assessed as follows. 2mg / ml of His-tagged recombinant human FcγR proteins were captured on an anti-penta-His sensor from Fortebio. Then, serially diluted tested antibodies (TY24118 or TY24122) were flowed over the sensor to allow binding, followed by a dissociation step in running buffer. The affinity of the antibodies to FcγR proteins was calculated by steady-state analysis. As shown in Figure Table E, TY24118 and TY24122 exhibited improved binding affinity to human FcγRIIa and FcγRIIb proteins, respectively, compared to the corresponding wild-type antibodies TY23310 and TY21242. TIFF2025509272000033.tif88170

[0129] Example 5: Binding of masked anti-CD137 antibodies to target T cells

[0130] Binding of masked anti-CD137 antibodies to target T cells was assessed as follows. Human and cynomolgus T cells were isolated from peripheral blood obtained from 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 or unstimulated naive T cells were incubated with serially diluted test antibodies along with the corresponding anti-CD4 and anti-CD8 antibodies to gate T cell subpopulations. Test antibody binding was detected with a fluorescently labeled anti-human IgGFc secondary antibody by FACS analysis. As shown in Figure 4A-D, only MM9 cleaved TY25368 showed positive staining on activated human T cells, cynomolgus T cells, mouse T cells, and rat T cells, consistent with induced CD137 expression in activated T cells. MM9 cleaved TY25368 showed no detectable binding to unstimulated naive T cells. Uncleaved TY25368 showed no detectable binding to activated or naive T cells. These results suggest that the masking peptide of the masked antibody TY25368 prevents its binding to target T cells and prevents unmasking by the target cell binding activity of TY25368 restored by protease cleavage.

[0131] Example 6: Ligand Inhibitory Activity of Masked Anti-CD137 Antibodies

[0132] The ligand inhibitory activity of anti-CD137 masked antibodies was evaluated as follows: 1 mg / ml recombinant human CD137 protein was coated onto an ELISA plate. Then, 2 mg / ml biotinylated recombinant human CD137 ligand was incubated with the CD137 pre-coated ELISA plate in the presence of serially diluted masked antibodies for 1 h at 37 °C. After washing, NeutrAvidin-HRP was added to the plate to detect the interaction between CD137 and its ligand. As shown in Figure 5, MMP9-cleaved TY25368 potently inhibited the interaction between CD137 and its ligand in a dose-dependent manner, with an IC50 of 4.46 nM. Uncleaved TY25368, similar to the isotype control antibody, showed no detectable inhibitory activity. These results indicate that TY25368, once cleaved, can inhibit ligand interaction with the CD137 receptor.

[0133] Example 7: Activation of cell signaling through CD137 by masked anti-CD137 antibodies

[0134] The stimulatory activity of anti-CD137 antibodies and their masked counterparts on CD137 receptor signaling was assessed using Jurkat-CD137-NFkB-luciferase reporter cells. Briefly, reporter cells were cultured in the presence or absence of CHO-K1-hFcγRIIb cells as a cross-linker (E:CL=20:1, "E" refers to Jurkat-CD137-NFkB-luciferase reporter cells, "CL" refers to CHO-K1-hFcγRIIb cells). Serially diluted test antibodies were added to the reporter cell system to assess their activity in stimulating downstream luciferase activity.

[0135] As shown in Figures 6A-6B and Table F, in the presence of CHO-K1-hFcγRIIb cells as crosslinkers, all anti-CD137 antibodies showed activation of CD137 signaling. MMP9-treated masked antibody TY25368 showed the strongest signal. The activity of the uncleaved masked antibody TY25368 was much weaker than the cleaved form. Other clinical anti-CD137 antibodies were also compared in this assay. Anti-CD137 antibodies AC1121 and AC1097 are described in WO 2019 / 036855 and US 2020 / 00369776, the contents of which are incorporated by reference herein in their entirety. Of note, in the absence of CHO-K1-hFcγRIIb cells as crosslinkers, only AC1121 showed activity. All other anti-CD137 antibodies were inactive, suggesting that CD137 agonist activity is crosslinking dependent. TIFF2025509272000034.tif108170

[0136] Example 8: Cross-linking dependent activation of human primary B cells by masked anti-CD137 antibodies

[0137] Stimulatory activity on CD137 receptor signaling by anti-CD137 antibodies and their masked counterparts was assessed as follows. Human primary B cells were isolated from peripheral blood collected from healthy donors. Jurkat-CD137-NFkB-luciferase reporter cells were cultured in the presence or absence of primary B cells as crosslinkers (E:CL=5:1 or 20:1). Serially diluted test antibodies were added to the reporter cell system and assessed for their ability to stimulate downstream luciferase activity. As shown in Figure 7, in the presence of human primary B cells as crosslinkers, at both E:CL ratios (5:1 or 20:1), cleaved TY25368-MMP9 showed the strongest CD137 activation signal. The activity of the uncleaved masked antibody TY25368 was much weaker than the cleaved form. Other clinical anti-CD137 antibodies were also compared in this assay.

[0138] Example 9: Activation of peripheral blood mononuclear cells by masked anti-CD137 antibodies with Staphylococcal enterotoxin A (SEA)

[0139] Anti-CD137 antibodies were evaluated to determine whether they could enhance human T cell activation by Staphylococcal enterotoxin A (SEA) peptide stimulation in human peripheral blood mononuclear cells (PBMCs) as follows. SEA, a superantigen, is known to activate a large proportion of human T cells by binding to MHC II expressed on the surface of antigen-presenting cells and the T cell receptor (TCR) expressed on the surface of T cells, and was therefore chosen to induce T cell activation in this study. Human PBMCs (2.0 × 10 5 T cells (100 / well of a 96-well plate) were isolated from two healthy donors (donor #102 and donor #142) and stimulated with a suboptimal concentration of SEA peptide (50 ng / mL). Then, serially diluted concentrations of anti-CD137 antibody and isotype control antibody were dispensed into the wells. After 4 days, replicate cell supernatants were collected and IL-2 was measured by ELISA as an endpoint of enhanced T cell activation.

[0140] As shown in Figure 8 and Table G, PBMCs from both donors demonstrated the highest levels of IL-2 cytokine secretion in the presence of cleaved TY25368 (i.e., TY25368 treated with MMP9). IL-2 cytokine secretion levels by PBMCs from both donors were very weak in the presence of uncleaved TY25368. Other clinical anti-CD137 antibodies (AC1121 and AC1097) were also tested in this assay. TIFF2025509272000035.tif100170

[0141] Example 10: Screening of anti-CD137 antibodies for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)

[0142] Anti-CD137 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. Serially diluted test antibodies were added to the ADCC reporter cell system to assess their ability to stimulate downstream luciferase activity. As shown in Figure 9, no ADCC reporter activity was detected on 293F-CD137 cells in the presence of MMP9-treated TY25368, untreated TY25369, or IgG1 isotype control antibody. In contrast, AC1121-IgG1 showed strong ADCC reporter activity against 293F-CD137 cells in a dose-dependent manner.

[0143] The masked anti-CD137 antibody TY25368 was screened for complement-dependent cytotoxicity (ADCC) as follows. Human T cells isolated from healthy donors were activated with anti-CD3 / anti-CD28 to induce high-level expression of CD137 and HLA-A / B / C. The activated T cells were cultured in the presence of normal human serum complement (NHSC). Then, serially diluted test antibodies were added to the assay system. As shown in Figure 10, no CDC activity was detected in the presence of MMP9-treated TY25368, untreated TY25368, or an IgG1 isotype control antibody. In contrast, the positive control antibody (i.e., mouse anti-human HLA-A / B / C) showed potent CDC activity against activated T cells in a dose-dependent manner.

[0144] Example 11: Antitumor Efficacy in CT26 Mouse Colon Tumor Model

[0145] BALB / c mice (female, 8-9 weeks old) were subcutaneously inoculated with CT26 mouse colon cancer cells. Tumors with a volume of approximately 80 mm 3At the time point, mice were randomly divided into four groups (n=8 per group) and treated with (a) vehicle control, (b) 1 mg / kg masked antibody TY25368, (c) 5 mg / kg anti-PD-1 antibody FG1225, or (d) 1 mg / kg masked antibody TY25368 and 5 mg / kg anti-PD-1 antibody FG1225. Treatments were administered twice weekly by intraperitoneal injection. Tumor growth was monitored twice weekly and reported as mean tumor volume ± standard error of measure (SEM) over time. As shown in Figure 11, TY25368 and FG1225 each showed partial efficacy as single agents. At 22 days after the start of treatment, tumor growth was inhibited by 48% in mice treated with TY25368 and 42% in mice treated with FC1225. In contrast, mice treated with the combination of TY25368 and FC1225 experienced 85% inhibition of tumor growth.

[0146] Example 12: Antitumor Effects in MC38 Mouse Colon Tumor Model

[0147] C57BL / 6 mice (female, 8-9 weeks old) were inoculated subcutaneously with MC28 mouse colon cancer cells. Tumors with a volume of approximately 80 mm 3At the time of reaching 100% CI, mice were randomly divided into eight groups (n=8 per group) and treated with (a) vehicle, (b) 5 mg / kg masked antibody TY25368, (c) 0.1 mg / kg anti-CTLA4 antibody TY21580, (d) 0.4 mg / kg masked 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. Treatments were administered twice weekly by intraperitoneal injection. Tumor growth was monitored twice weekly and reported as mean tumor volume ± SEM over time. As shown in Figure 12, TY21580 and TY22404 each showed partial efficacy as single agents. Three out of eight mice in the TY21580 single agent group showed no tumors. Tumor growth was inhibited in one of eight mice treated with TY22404 as a single agent. Combination treatment with TY25368 and TY21580, or TY25368 and TY22404, significantly improved the antitumor efficacy. See Figure 12.5. Eight mice treated with TY25368 and TY21580 were tumor-free. All eight mice treated with TY25368 and TY22404 were tumor-free.

[0148] Example 13: Pharmacokinetic (PK) and Pharmacodynamic (PD) Studies of TY25368 in Cynomolgus Monkeys

[0149] Cynomolgus monkeys were administered TY25368 30 mg / kg or TY25368 100 mg / kg intravenously once weekly for two weeks. Plasma concentrations of antibody TY25368 (intact (i.e., uncleaved masked antibody) and total (i.e., both uncleaved and cleaved forms) were measured at different time points using an ELISA assay. The results are shown in Table H below. Peripheral T lymphocytes were also profiled by FACS analysis, but no significant changes were observed (data not shown). The monkeys tolerated the antibody administration well and no clinical symptoms were observed during the study. TIFF2025509272000036.tif50170Cmax = maximum (peak) serum concentration, Tmax = time to reach Cmax, AUC = area under the curve representing the change in drug concentration in plasma as a function of time, T1 / 2 = half-life, Vd = total systemic drug / plasma drug concentration, Cl = clearance. TIFF2025509272000037.tif45170

[0150] As shown in Figure 13 and Table H, TY25368 exhibited a linear PK profile after the first dose in the dose range between 30 mg / kg and 100 mg / kg for both the whole and intact forms. After the second 30 mg / kg dose, TY25368 had a faster clearance and shorter T1 / 2. Without being bound by theory, such effects may be due to anti-drug antibodies (ADAs) induced by the treatment. Comparing the whole and intact forms after dosing, high and similar drug concentrations and drug exposures were observed, indicating that the masked antibody ty25368 was stable in peripheral blood in monkeys. Thus, preclinical toxicology studies have demonstrated that TY25366 and TY25368 were well tolerated in monkeys, had normal pharmacokinetic behavior, and minimal activation within the circulation.

[0151] Example 14: Pharmacokinetic (PK) studies of TY21242, TY24118, and TY25366 in mice

[0152] Pharmacokinetic studies were performed in BALB / c female mice bearing CT26 tumors. Mice were randomly divided into four groups (n=4 per group) and intraperitoneally injected with TY25368, TY25366, TY21242, TY24118, or TY24122 at 5 mg / kg. Blood samples (approximately 50 μl per sample) were collected at 3, 6, 24, 48, 96, 168, and 336 hours after dosing. Blood concentrations of TY25368, TY25366, TY21242, TY24118, and TY24122 were measured by ELISA using anti-human IgG Fc antibody as a capture agent and HRP-conjugated anti-human IgG (Fab specific) antibody as a detection agent. A second set of ELISA assays using specific anti-idiotypic antibodies as the capture agent and HRP-conjugated anti-human IgG (Fab-specific) antibodies as the detection agent were performed to detect the active (i.e., unmasked) forms of TY25368 and TY25366. A description of each antibody is provided in Table I. TIFF2025509272000038.tif112170

[0153] TY25368 had a half-life of 98 hours and a drug concentration of approximately 6.48 μg / ml at 336 hours. TY25366 had a half-life of 28 hours and a drug concentration of approximately 0.13 μg / ml at 336 hours. In comparison, the parent antibody TY21242 had a half-life of 74 hours and a drug concentration of approximately 3.09 μg / ml at 336 hours. TY24118 had a half-life of 77 hours and a drug concentration of approximately 2.50 μg / ml at 336 hours. TY24122 had a half-life of 79 hours and a drug concentration of approximately 3.74 μg / ml at 336 hours. TY25368 had a slower clearance time and a longer half-life than the parent antibody, whereas TY25366 had a much faster clearance time and a shorter half-life than the parent antibody. 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.

[0154] Accordingly, preclinical toxicology studies have demonstrated that TY25366 and TY25368 are well tolerated in mice, have normal pharmacokinetic behavior, and are minimally activated in the circulation.

[0155] Example 15: Activation of cell signaling through CD137 by anti-CD137 antibodies containing Fc mutations

[0156] Fc mutations in Table J that could potentially affect cross-linking were introduced into TY23310 or TY21242 to generate TY24117, TY24118, TY24119, TY24120, TY24121, and TY24122. TIFF2025509272000039.tif55170 a See Chu et al. (2008) Mol Immunol. 45(15):3926-33. b See Mimoto et al. (2013) Protein Eng Des Sel. 26(10):589-98.

[0157] Anti-CD137 antibodies were transiently expressed in HEK293F cells and purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The agonist activity of anti-CD137 antibodies was compared in a Jurkat / NFkB reporter gene assay. Briefly, human embryonic kidney 293T cells were transiently transfected with plasmids expressing the human CD137 receptor, an NFκB firefly luciferase reporter, and a control Renilla luciferase reporter construct. Cells were incubated with test antibodies in the presence or absence of (a) human CHO-K1-hFcγRIIb (also called CHO-K1-FcγRIIb) or (b) mouse CHO-K1-mFcγRIIb. The relative level of CD137 signaling activation was then measured by firefly luciferase activity after normalization with Renilla luciferase activity. As shown in Figures 14A-14C, in the presence of CHO-K1-FcγRIIb, both antibodies tested showed higher CD137 agonist activity than TY23310 for wild-type IgG1 or TY21242 for wild-type IgG4. With respect to CHO-K1-mFcγRIIb as a crosslinker, no activity enhancement was observed or it was lower than the activity enhancement observed in the human system. In the absence of a crosslinker, none of the antibodies showed cell signaling through CD137.

[0158] Example 16: Activation of peripheral blood mononuclear cells by anti-CD137 antibodies with Staphylococcal enterotoxin A (SEA)

[0159] The anti-CD137 antibodies generated in Example 15 were evaluated as follows to determine whether they could enhance human T cell activation by Staphylococcal Enterotoxin A (SEA) peptide stimulation in human peripheral blood mononuclear cells (PBMCs). Fresh human PBMCs were incubated with 50 ng / mL SEA and serially diluted concentrations of anti-CD137 antibodies (in soluble form or immobilized on a solid support) for 96 hours, after which cell culture supernatants were collected to measure IL-2 levels. As shown in Figure 15, the anti-CD137 antibodies TY24118 and TY24122 showed better agonist activity than TY24117, TY24118, TY24119, TY24120, TY24121 and TY24122.

[0160] Example 17: Affinity of TY25368 and TY25368-MMP9 to human CD137, cynomolgus monkey CD137, mouse CD137, and rat CD137.

[0161] The binding affinity of TY25368 and its activated form (termed TY25368-MMP9) to recombinant CD137 of various species was evaluated by surface plasmon resonance (SPR). Briefly, anti-human IgG (Fc) antibody (Cytiva, Cat. No. BR-1008-39) was immobilized on a CM5 chip by amide coupling according to the instructions of the amine coupling kit (Cytiva, Cat. No. BR-1000-50). The final response of the immobilization level was approximately 5000 RU (relative units). TY25368 and TY25368-MMP9 were each diluted to 3 μg / mL in 1× HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA·2Na, 0.005% (v / v) surfactant P20, Ph 7.4) and then injected into the system at a flow rate of 10 μL / min for 30 s to immobilize. CD137 antigen was serially diluted in 1x HBS-EP buffer and flowed over the CM5 chip with immobilized test antibody at a flow rate of 30 μL / min for 300 s. After binding, 1x HBS-EP buffer was injected over the surface at a flow rate of 30 μL / min for an additional 300 s to test the antigen dissociation rate. For regeneration of the CM5 sensor chip, 3 M magnesium chloride solution was used to remove residual antibody / antigen. As shown in Table K, TY25368 has low binding affinity (KD>1000 nM) for human CD137, cynomolgus monkey CD137, mouse CD137, and rat CD137. In contrast, activated TY25368-MMP9 was found to bind with high affinity to human CD137 and cynomolgus monkey CD137 (KD of 3.19 nM and 4.23 nM, respectively). Activated TY25368-MMP9 was also found to bind with low affinity to mouse CD137 and rat CD137 (KD of 27.54 nM and 42.37 nM, respectively). TIFF2025509272000040.tif95170

[0162] Example 17: Antitumor effect of TY25368 in mouse EMT6 breast cancer model

[0163] BALB / c mice (4 groups, n = 8 in each group, female, 8–9 weeks old) were subcutaneously inoculated with EMT6 (ATCC) mouse mammary carcinoma cells. When tumors were established (i.e., tumor volume was approximately 120 mm), 3 At the time point when tumor volume reached 100 mg / kg, mice were treated with (a) vehicle, (b) 3 mg / kg of masked anti-CD137 antibody TY25368, (c) 1 mg / kg of TY25368, or (d) 0.3 mg / kg of TY25368. Treatments were administered intraperitoneally twice weekly. Tumor growth was monitored twice weekly and reported as the mean tumor volume ± standard error over time. As shown in Figure 16, TY25368 demonstrated a dose-dependent antitumor effect in the EMT6 mouse allograft tumor model. Tumor regression was observed in all mice receiving TY25368 1 mg / kg and TY25368 3 mg / kg.

[0164] Example 18: Antitumor effects of TY24118, TY24122, TY25366, and TY25368 in H22 mouse liver cancer model

[0165] BALB / c mice (5 groups, n = 8 per group, female, 8-9 weeks old) were subcutaneously inoculated with H22(CCTCC) mouse hepatoma cells. When tumors were established (i.e., tumor volume was approximately 90 mm), 3 When tumor volume reached 100 mg / kg, 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. (Antibody descriptions are provided in Table I above.)) Treatments were administered by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly and reported as mean tumor volume ± standard error over time. As shown in Figure 17, the unmasked parental anti-CD137 antibodies TY24118 and TY24122, and the masked anti-CD137 antibodies TY25366 and TY25368, both at 5 mg / kg, demonstrated similar efficacy.

[0166] Example 19: Antitumor effects of TY24118, TY24122, TY25366, and TY25368 in a mouse CT26 colon cancer model

[0167] BALB / c mice (5 groups, n = 8 per group, female, 9–10 weeks old) were inoculated subcutaneously with CT26 (SIBS) mouse colon carcinoma cells. When tumors were established (i.e., tumor volume was approximately 60 mm), 3 When tumor volume reached 100 mg / kg, mice were treated with (a) vehicle, (b) TY24118, (c) TY24122, (d) TY25366, or (e) TY25368. (Antibody descriptions are provided in Table I above.) Antibodies were administered at 5 mg / kg or 1 mg / kg by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly and reported as mean tumor volume ± standard error over time. As shown in Figures 18A and 18B, the unmasked parental anti-CD137 antibodies TY24118 and TY24122 demonstrated similar efficacy at both high (5 mg / kg) and low (1 mg / kg) doses. The masked anti-CD137 antibody TY25368 demonstrated similar efficacy to TY24118 and TY24122 at both high (5 mg / kg) and low (1 mg / kg) doses. The masked anti-CD137 antibody TY25366 was less potent than TY24118 and TY24122 at both high (5 mg / kg) and low (1 mg / kg) doses.

[0168] Typical conclusion

[0169] TY25368 is an Fc-enhanced masked anti-CD137 with broad species cross-reactivity. TY25368 has high masking efficiency, is conditionally activated, and binds strongly to the CD137 costimulatory receptor on activated T cells. TY25368 showed stronger FcγR-dependent stimulation of CD137 signaling than urelumab. TY25368 showed stronger anti-CD137 agonist activity than urelumab on T cell activation in the presence of primary stimulatory signals, although the activity of masked TY25368 was much lower. TY25368 showed potent antitumor activity as a single agent and cooperated with other immune checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4 to enhance antitumor efficacy. TY25368 was well tolerated in rats and cynomolgus monkeys in nonclinical toxicity studies, showed normal pharmacokinetic behavior, and showed minimal activation during circulation.

[0170] Example 20: Antitumor Efficacy of TY25368 in Combination with TY27151 in a Murine MC38-HER2-B7H3 Colon Adenocarcinoma Model

[0171] C57BL / 6-hCD3e mice (n=6 per group, female, 9 weeks old) were inoculated subcutaneously with MC38-HER2-B7H3 murine colon adenocarcinoma cells. C57BL / 6-hCD3e mice are derived from C57BL / 6 and carry the human CD3E gene. Such mice express hCD3ε on T cells. The MC38-HER2-B7H3 cell line is derived from the MC38 cell line and engineered to overexpress human HER2 ("hHER2") and human B7H3 ("hB7H3"). When tumors were established (i.e., tumor volume was (~105 mm 3), mice were treated with (a) vehicle, (b) TY25368, (c) TY27151, or (d) TY25368 and TY27151. Antibodies were administered by intraperitoneal injection twice weekly at 5 mg / kg each. A description of TY25368 is provided in Table I above. TY27151 is a bispecific T cell engager (TCE) construct that binds HER2 and CD3. Both the anti-CD3 and anti-HER2 arms of TY27151 consist of a masking moiety containing a protease cleavage site and a linkage unit. The amino acid sequences of the heavy and light chains of TY27151 are shown in Table L below. The masking peptide of TY27151 is shown in bold. The masking sequence is underlined and the linkage unit is italicized. TY27151 is composed of three polypeptide chains: an anti-HER2 antibody heavy chain, an anti-HER2 antibody light chain, and an ScFv-Fc fusion polypeptide in which the C-terminus of the ScFv is fused to the N-terminus of the Fc domain. The anti-HER2 antibody heavy chain and the anti-HER2 antibody light chain combine 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 polypeptide of the anti-CD3 ScFv-Fc fusion dimerize to form a TCE. See, e.g., FIG. 20 for a schematic diagram of TY27151. Additional details regarding TY27151 are described in WO 2022 / 170740, the contents of which are incorporated herein by reference in their entirety. TIFF2025509272000041.tif135170* Masking sequences are in bold and underlined. Linkage units are shown in bold italics.

[0172] Once tumors were identified, treatment was initiated with vehicle, TY25368 (5 mg / kg), HER2xCD3 double-masked bispecific antibody TY27151 (5 mg / kg), or their combination by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly and reported as mean tumor volume ± standard error over time. As shown in Figure 19A, 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% and tumor disappearance in 2 of 8 mice on day 22. Combination treatment with TY25368 and TY27151 significantly improved antitumor efficacy, with a TGI of 85% and tumor disappearance in 3 of 8 mice on day 22. Tumor growth curves for individual mice in each treatment group (a), (b), (c), and (d) are shown in Figure 19B.

[0173] The present invention has been described in terms of specific embodiments discovered or proposed by the inventor as including the preferred modes for carrying out the invention. Those skilled in the art will recognize in light of this disclosure that numerous modifications and variations can be made to the specific embodiments exemplified without departing from the intended scope of the invention. For example, codon redundancy allows for changes to be made to the underlying DNA sequence without affecting the protein sequence. Furthermore, considerations of biological functional equivalence allow for changes to be made to the protein structure without affecting the biological action in terms of type or amount. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A masked antibody comprising a masking peptide (MP) and an antibody that binds to human CD137, The antibody comprises (a) a heavy chain comprising a heavy chain variable region (VH) and an Fc region, and (b) a light chain comprising a light chain variable region (VL); the MP is linked to the N-terminus of the VL, and the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7; VH comprises CDR-H1 represented by TGGVGVG (SEQ ID NO: 36), CDR-H2 represented by LIDWADDKYYSPSLKS (SEQ ID NO: 37), and CDR-H3 represented by GGSDTVIGDWFAY (SEQ ID NO: 38), and VL comprises CDR-L1 represented by RASQSIGSYLA (SEQ ID NO: 39), CDR-L2 represented by DASNLET (SEQ ID NO: 40), and CDR-L3 represented by QQGYYLWT (SEQ ID NO: 41); and The Fc region is (i) G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; 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 or a variant of a human IgGl Fc region comprising substitutions selected from the group consisting of: P271G; G237D, P238D, P271G, and A330R; E233D, H268D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; E233D, G237D, P238D, H268D, P271G, and A330R; T437R; K248E; and T437R and K248E, wherein amino acid numbering is according to the EU index; or (ii) G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; 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 a variant of a human IgG4 Fc region comprising substitutions selected from the group consisting of: G237D, P238D, P271G, and A330R; E233D, H268D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; E233D, G237D, P238D, H268D, P271G, and A330R; T437R; K248E; and T437R and K248E, wherein amino acid numbering is according to the EU index; Masked antibodies.

2. The masked antibody according to claim 1, wherein the LU includes a first cleavage site.

3. The first cleavage site is selected from the group consisting of 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, and ADAM12.

3. The masked antibody of claim 2, wherein the protease cleavage site is a protease cleavage site of a protease selected from the group consisting of ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14 and TACE.

4. The aforementioned LU is further a first linker (L １ The masked antibody according to claim 2, comprising a second cleavage site and / or a second cleavage site.

5. (a) The second cutting portion is L １ (b) The C-terminus of and / or (b) the second cleavage site is 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, ADA The masked antibody according to claim 4, wherein the masked antibody is a protease cleavage site of a protease selected from the group consisting of M10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

6. The aforementioned LU further comprises a second linker (L ２ A masked antibody according to claim 4, comprising )

7. The masked antibody according to claim 1, wherein the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 8, 10-14, and 16.

8. A masked antibody comprising a masking peptide (MP) and an antibody that binds to human CD137, The antibody comprises (a) a heavy chain comprising a heavy chain variable region (VH), and (b) a light chain comprising a light chain variable region (VL); the MP is linked to the N-terminus of the VL, and the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, and the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 8, 10-14, and 16; VH comprises CDR-H1 represented by TGGVGVG (SEQ ID NO: 36), CDR-H2 represented by LIDWADDKYYSPSLKS (SEQ ID NO: 37), and CDR-H3 represented by GGSDTVIGDWFAY (SEQ ID NO: 38), and VL comprises CDR-L1 represented by RASQSIGSYLA (SEQ ID NO: 39), CDR-L2 represented by DASNLET (SEQ ID NO: 40), and CDR-L3 represented by QQGYYLWT (SEQ ID NO: 41); Masked antibodies.

9. The antibody, (a) 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, minibodies, and diabodies; or (b) a full-length antibody comprising an Fc region, Optionally, the Fc region is a human IgG Fc region or a variant thereof; Optionally, the human IgG Fc region or 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; Further optionally, the Fc region comprises: (i) G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; 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 or a variant of a human IgGl Fc region comprising substitutions selected from the group consisting of: P271G; G237D, P238D, P271G, and A330R; E233D, H268D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; E233D, G237D, P238D, H268D, P271G, and A330R; T437R; K248E; and T437R and K248E, wherein amino acid numbering is according to the EU index; or (ii) G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; 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; G23 A full-length antibody that is a variant of the human IgG4Fc region, including substitutions selected from the group consisting of 7D, P238D, P271G, and A330R; E233D, H268D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; E233D, G237D, P238D, H268D, P271G, and A330R; T437R; K248E; and T437R and K248E, wherein the amino acid numbering follows the EU index. The masked antibody according to claim 8.

10. A masked antibody described in any one of claims 1 to 9, wherein the MP further comprises an N-terminal unit (NU) linked to the N-terminus of the MU.

11. The masked antibody described in claim 10, wherein the N-terminal unit (NU) is (a) approximately 1 to 10 amino acid residues in length and / or (b) comprises E or EVGSY (sequence number 77).

12. The masked antibody of any one of claims 1 to 9, wherein the MP comprises any one of SEQ ID NOs: 17 to 35. (a) the VH comprises SEQ ID NO: 52 and the VL comprises SEQ ID NO: 53; or (b) the VH comprises SEQ ID NO: 52 and the VL comprises SEQ ID NO: 58; A masked antibody according to any one of claims 1 to 9.

14. (a) A variant of the human IgG1 Fc region comprising S267E and L328F substitutions, or (b) a variant of a human IgG4 Fc region comprising S267E and L328F substitutions; The masked antibody of any one of claims 1 to 7 and 9, wherein the amino acid numbering is according to the EU index.

15. (a) The Fc region is a variant of the human IgG1 Fc region containing SEQ ID NO: 113 or SEQ ID NO: 114, or (b) The Fc region is a variant of the human IgG4 Fc region containing SEQ ID NO: 117 or SEQ ID NO:

118. The masked antibody of claim 14.

16. The MP set forth in SEQ ID NO: 34, the VH set forth in SEQ ID NO: 52, the VL set forth in SEQ ID NO: 53, and (a) a human IgG1 Fc region variant comprising S267E and L328F substitutions, or (b) a human IgG4 Fc region variant comprising S267E and L328F substitutions; A masked antibody according to claim 12, comprising, wherein the amino acid number follows the EU index.

17. (a) The heavy chain includes sequence number 94 or 95, and the light chain includes sequence number 96, or (b) the heavy chain comprises SEQ ID NO: 92 or 93, and the light chain comprises SEQ ID NO: 96; The masked antibody of claim 16.

18. One or more polynucleotides encoding a masked antibody according to any one of claims 1 to 9.

19. 20. A recombinant vector comprising one or more polynucleotides of claim 18.

20. A host cell comprising the recombinant vector according to claim 19.

21. 21. A method for producing a masked antibody, comprising culturing the host cell of claim 20 under appropriate conditions to cause expression of the masked antibody, and recovering the masked antibody.

22. A pharmaceutical product comprising a masked antibody according to any one of claims 1 to 9, for treating cancer in a subject.

23. The pharmaceutical product according to claim 22, wherein the cancer is a solid tumor.

24. The pharmaceutical product according to claim 23, wherein the solid tumor is breast cancer, liver cancer, colorectal cancer, or colon cancer.

25. The pharmaceutical composition of claim 22, further comprising: (a) an anti-PD-1 antibody; (b) an anti-CTLA4 antibody; (c) a bispecific antibody that binds HER2 and CD3; or (d) a bispecific T-cell engager (TCE) that targets CD3 and an antigen expressed on the surface of solid tumor cancer cells.

26. (a) The antigen expressed on the surface of the solid tumor cancer cells is HER2, and / or (b) The pharmaceutical of claim 25, wherein the TCE comprises three polypeptide chains, 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 domain fusion in which the C-terminus of the scFv is fused to the N-terminus of the Fc domain, the first and second polypeptide chains associate to form a HER-2 binding arm, the third polypeptide chain binds to CD3, and the Fc domain of the anti-HER2 binding arm and the Fc of the third polypeptide chain dimerize to form the TCE.

27. 27. The pharmaceutical composition of claim 26, 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.

28. A kit comprising a masked antibody according to any one of claims 1 to 9 for use in a method of treating cancer in an individual.

29. The kit of claim 28, further comprising (i) an anti-PD-1 antibody, (ii) an anti-CTLA4 antibody, (iii) a bispecific antibody that binds HER2 and CD3, or (iv) a bispecific T-cell engager (TCE) that targets CD3 and an antigen expressed on the surface of solid tumor cancer cells.

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