IL-2 procytokine antibody fusion protein

Abstract

Provided is an activatable proprotein homodimer comprising two separate but identical polypeptide chains, each chain comprising an antigen-binding fragment (Fab) region that specifically binds human PD-1, human PD-L1, or human B7H3, a hinge / Fc domain, a linker, an IL-2 protein, a protease-cleavable linker, and an IL-2Rα protein. Related pharmaceutical compositions and methods of use thereof are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 393,150, filed July 28, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing Statement The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference. The XML file containing the Sequence Listing XML is named PRVA_015_01WO_ST26.xml. The XML file is approximately 286,496 bytes, was created on July 27, 2023, and has been submitted electronically via the USPTO Patent Center. [Background technology]

[0003] background Technical Field The present disclosure relates to an activatable proprotein homodimer comprising two separate but identical polypeptide chains, each chain comprising an antigen-binding fragment (Fab) region that specifically binds human PD-1, human PD-L1, or human B7H3, a hinge / Fc domain, a linker, an IL-2 protein, a protease-cleavable linker, and an IL-2Rα protein. Related pharmaceutical compositions and methods of use thereof are also included. Description of related fields Interleukin-2 (IL-2) immunotherapy has proven useful in the treatment of cancer, including malignant melanoma and renal cell carcinoma, among others. Programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitor therapy enhances antitumor T cell responses and mediates antitumor activity (Dermani et al., J Cell Physiol. 234:1313-1325, 2019). However, there are certain problems associated with most IL-2 therapies. For example, current forms of IL-2 therapy have a short half-life in the circulation and inhibit the production of immunosuppressive regulatory T cells, or T reg (See, e.g., Arenas-Ramirez et al., Trends in Immunology. 36: 763-777, 2015). Furthermore, the effects of IL-2 therapy are primarily systemic rather than localized to target tissues, resulting in numerous severe side effects, such as respiratory problems, nausea, hypotension, loss of appetite, confusion, serious infections, seizures, allergic reactions, cardiac problems, renal failure, and vascular leak syndrome. Nevertheless, IL-2 therapy can be effective, and strategies exist to address these and other drawbacks (see, e.g., WO2021 / 011353). However, improvements to such strategies remain a need in the art. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2021 / 011353 [Non-patent literature]

[0005] [Non-Patent Document 1] Dermani et al., J Cell Physiol. 234:1313-1325, 2019 [Non-patent document 2] Arenas-Ramirez et al., Trends in Immunology. 36: 763-777, 2015 Summary of the Invention [Problem to be solved by the invention]

[0006] Embodiments of the present disclosure represent such an improvement by providing anti-PD-1 / PD-L1 activatable proproteins (procytokines), including IL-2, that can be specifically targeted to and activated in the tumor microenvironment (TME). [Means for solving the problem]

[0007] Abstract An embodiment of the present disclosure is an activatable proprotein homodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, in N-terminal to C-terminal orientation, an antigen-binding fragment (Fab) region that specifically binds to human PD-1 or human PD-L1 or human B7H3, a hinge / Fc domain, a first linker, an IL-2 protein, a second linker, and an IL-2Rα protein, wherein the hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, the IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, and the IL-2Rα of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the binding is in vitro or in vivo to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell. It comprises an activatable proprotein homodimer that masks the binding site of the IL-2 protein(s) that would otherwise bind in vivo, and the second linker is a cleavable linker.

[0008] In some embodiments, the Fab region specifically binds human PD-1, and optionally comprises a Fab region from an anti-PD-1 antibody selected from nivolumab, pembrolizumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, MGA012, AMP-22, and AMP-514. In some embodiments, the Fab region specifically binds human PD-1, and a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 1; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 2; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO:3; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO:4; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO:5; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO:6; or a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO:7; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO:8; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO:9; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO:10; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 11; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 12; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 13; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 14; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 15; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 16; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 17; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 18; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 19; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 20; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 21; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 22; or a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 23; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 24; Includes.

[0009] In some embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, 21 and 23, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24, respectively.

[0010] In some embodiments, the Fab region specifically binds human PD-L1, and optionally comprises a Fab region from an anti-PD-L1 antibody selected from atezolizumab, avelumab, and durvalumab. In some embodiments, the Fab region specifically binds human PD-L1, and a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 25; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 26; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 27; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 28; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 29; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 30; or a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 31; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 32; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 33; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 34; a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 35; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 36; or a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2 and VHCDR3 regions set forth in SEQ ID NO: 37; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2 and VLCDR3 regions set forth in SEQ ID NO: 38; Includes.

[0011] In some embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35 and 37, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36 and 38, respectively.

[0012] In some embodiments, the Fab region specifically binds to human B7H3, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 261, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 262, respectively.

[0013] In some embodiments, the Fc domain comprises a CH2 domain, a CH3 domain, or a CH2CH3 domain of an immunoglobulin, optionally wherein the immunoglobulin is from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. In some embodiments, the hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from Table F1, and the Fc domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from Table F1. In some embodiments, the Fc domain is a modified Fc domain that does not bind, or does not substantially bind, to FcγR and retains normal or substantially normal binding to FcRn. In some embodiments, the modified Fc domain comprises a modified IgG1 CH2 domain with L234A / L235A (“LALA”) mutations and / or P329A or P329G mutations (EU numbering).

[0014] In some embodiments, the IL-2 protein comprises, consists of, or consists essentially of an amino acid sequence selected from Table S1, optionally containing a C145X (X is any amino acid) or C145S substitution as defined by SEQ ID NO:68, an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to amino acids 21-153 of SEQ ID NO:68 (full-length wild-type human IL-2). In some embodiments, the IL-2 protein comprises, consists of, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:69 (mature human IL-2 with a C125S substitution), optionally wherein the IL-2 protein retains the S125 residue as defined by SEQ ID NO:69. In some embodiments, the IL-2 protein contains one or more substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C as defined by SEQ ID NO:69. In some embodiments, the IL-2 protein of the first polypeptide forms a disulfide bond with the IL-2Rα protein of the second polypeptide, and the IL-2Rα protein of the first polypeptide forms a disulfide bond with the IL-2 protein of the second polypeptide, optionally via one or more of the cysteines described above. In some embodiments, the IL-2 protein comprises one or more amino acid substitutions at positions 69, 74, and / or 128 as defined by SEQ ID NO:69, optionally wherein the one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO:69.

[0015] In some embodiments, the IL-2 protein comprises one or more amino acid substitutions at positions T3, R38, F42, K43, Y45, E61, E62, E68 and / or L72 as defined by SEQ ID NO: 69, optionally the one or more amino acid substitutions are a combination thereof, optionally F42A, Y45A and L72G; R38K, F42Q, Y45N, E62L and E68V; R38K, F42Q, Y45E and E68V; R38A, F42I, Y45N, E62L and E68V; R38K, F42K, Y45R, E62L and E68V; R38K, F42I, Y45R and T3A, including combinations selected from R38A, F42A, Y45A and E62A; R38A, R38D and R38K; F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K and F42I; K43E; Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K; E61S; E62A and E62L; E68A and E68V; and L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In some embodiments, the IL-2 protein comprises, consists of, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 84 or 85 (human IL-2 mature (26-153)), optionally wherein the IL-2 protein comprises or retains the R38D, K43E, and C125S substitutions of SEQ ID NO: 85.

[0016] In some embodiments, the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence selected from Table S2, optionally at least 80, 85, 90, 95, 98, or 100% of amino acids 22-187 of SEQ ID NO: 86 (full-length wild-type human IL-2Rα), and optionally the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% of SEQ ID NO: 90, including when the IL-2Rα protein retains a D6R and / or E29K substitution. In some embodiments, the IL-2Rα protein comprises one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C, and / or a K38S substitution as defined by SEQ ID NO: 88 (the Sushi1-Sushi2 domains of human IL-2Rα). In some embodiments, the IL-2Rα protein of the first polypeptide forms a disulfide bond with the IL-2 protein of the second polypeptide, and the IL-2Rα protein of the second polypeptide forms a disulfide bond with the IL-2 protein of the first polypeptide, optionally via one or more cysteine ​​pairs selected from one or more of the above-mentioned cysteines and one or more cysteines in the IL-2 protein, optionally selected from IL2-K35C and IL2Rα-D4C, IL2-R38C and IL2Rα-D6C, IL2-R38C and IL2Rα-H120C, IL2-T41C and IL2Rα-I118C, IL2-F42C and IL2Rα-N27C, IL2-E61C and IL2Rα-K38C, IL2-E61C and IL2Rα-S39C, and IL2-V69C and IL2Rα-L42C. In some embodiments, the first and second IL-2Rα proteins comprise an alanine substitution at positions 49 and / or 68 as defined by SEQ ID NO:88.

[0017] In some embodiments, the hinge of the first polypeptide forms at least one or two disulfide bonds with the hinge of the second polypeptide. In some embodiments, the first linker is a non-cleavable or stable linker, wherein the cleavable linker comprises a protease cleavage site, optionally selected from Table S3. In certain embodiments, the first linker is a non-cleavable or stable linker 7 or less amino acids in length (or 1, 2, 3, 4, 5, 6, 7 amino acids in length), wherein the cleavable linker comprises a protease cleavage site, optionally selected from Table S3, e.g., SEQ ID NO: 93. In a specific embodiment, the first linker is a non-cleavable or stable linker, e.g., a 4-amino acid stable linker, e.g., GGGS (SEQ ID NO: 188), wherein the cleavable linker comprises a protease cleavage site, optionally selected from Table S3, e.g., SEQ ID NO: 93.

[0018] In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of a metalloprotease, a serine protease, a cysteine ​​protease, and an aspartic acid protease. In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A, and cathepsin B. In some embodiments, the first linker and / or the second linker is about 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3 amino acids in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acids in length.

[0019] In some embodiments, the Fab comprises SEQ ID NO: 3 (VH) and a human IgG1 CH1 domain and SEQ ID NO: 4 (VL) and a CL domain (kappa); the Fc domain comprises an IgG1 hinge of SEQ ID NO: 42, a modified human IgG1 CH2 domain of SEQ ID NO: 57, and a human IgG1 CH3 domain of SEQ ID NO: 58; the first linker is a 4-amino acid stable linker of SEQ ID NO: 188; the IL-2 protein comprises SEQ ID NO: 84 or 85, optionally with R38D, K43E, and C125S mutations; the second linker is a protease-cleavable linker of SEQ ID NO: 93; and the IL-2Rα protein comprises SEQ ID NO: 88 or 90, optionally with D6R and E29K mutations.

[0020] In certain activatable proprotein homodimers, cleavage of the second linker, optionally with a protease, exposes binding site(s) of the IL-2 protein that binds in vitro or in vivo to the IL-2Rβ / γc chain present on the surface of an immune cell. In some embodiments, the immune cell is selected from one or more of a T cell, a B cell, a natural killer cell, a monocyte, and a macrophage.

[0021] In some embodiments, the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S4 (chains 1 and 2), and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to the corresponding sequence from Table S4 (chains 3 and 4);

[0022] the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 138 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 139; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 140 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 141; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 142 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 143; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 146 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 147; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 148 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 149; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 152 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 153; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 154 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 155; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 156 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 157; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 158 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 159; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:209 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:210; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:211 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:212; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:213 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:214; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:215 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:216; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:217 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:218; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:219 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:220; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:221 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:222; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:223 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:224; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:225 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:226; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:227 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:228; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:229 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:230; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:231 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:232; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:233 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:234; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:235 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:236; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:237 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:238; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:239 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:240; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:241 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:242; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:243 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:244; the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:245 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:246; or the first polypeptide and the second polypeptide comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:247 and a VL / CL region polypeptide at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO:248; This includes cases.

[0023] Certain activatable proprotein homodimers described herein are substantially in homodimeric form in physiological solution or under physiological conditions, optionally in vivo conditions.

[0024] Also included are one or more recombinant nucleic acid molecules encoding the activatable proprotein homodimers described herein. In some embodiments, a first recombinant nucleic acid molecule encodes the VH / CH1 region of the Fab region, the hinge / Fc domain, the first linker, the IL-2 protein, the second linker, and the IL-2Rα protein, and a second nucleic acid molecule encodes the VL / CL region of the Fab region. Also included are one or more vectors comprising one or more recombinant nucleic acid molecules described herein. Certain embodiments include host cells comprising one or more recombinant nucleic acid molecules described herein or one or more vectors described herein.

[0025] Certain embodiments include a method of producing an activatable proprotein, comprising culturing a host cell described herein under culture conditions suitable for expression of an activatable proprotein homodimer, and isolating the activatable proprotein from the culture.

[0026] Also included are pharmaceutical compositions comprising the activatable proprotein homodimers described herein and a pharmaceutically acceptable carrier.

[0027] Certain embodiments relate to methods of treating a disease in a subject and / or enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0028] In some embodiments, the disease is cancer, eg, a cancer that expresses or overexpresses PD-L1. In some embodiments, the cancer is a primary or metastatic cancer and is selected from one or more of melanoma (optionally metastatic melanoma), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0029] In some embodiments, after administration, the activatable proprotein homodimer is activated via protease cleavage, exposing a binding site(s) of the IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of immune cells in cancer cells or tissues, or in the tumor microenvironment (TME), in vitro or in vivo, thereby generating an activated protein. In some embodiments, the activated protein binds via the IL-2 protein to the IL-2Rβ / γc chain present on the surface of immune cells in vitro or in vivo. In some embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0030] In some embodiments, administration and activation of the activatable proprotein increases an anti-cancer immune response in a subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to a control. In some embodiments, administration and activation of the activatable proprotein increases cancer cell killing in a subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to a control.

[0031] In some embodiments, the pharmaceutical composition is administered to the subject by parenteral administration, hi some embodiments, the parenteral administration is intravenous administration.

[0032] Certain embodiments include the use of a pharmaceutical composition described herein in the preparation of a medicament for treating a disease, optionally a cancer (e.g., a PD-L1-expressing or overexpressing cancer) in a subject and / or for enhancing an immune response in a subject. Certain embodiments include a pharmaceutical composition described herein for use in treating a disease, optionally a cancer (e.g., a PD-L1-expressing or overexpressing cancer) in a subject and / or for enhancing an immune response in a subject. [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1 shows an exemplary structure of a single polypeptide (IgG-proIL-2 motif) that forms a homodimer (not shown) with a second polypeptide of the same structure. The IgG-proIL-2 motif is composed of a high-affinity neutralizing antibody (e.g., no FcγR binding, but intact FcRn binding) and an IL-2 procytokine module.

[0034] [Figure 2] Figure 2 shows an exemplary structure of an activatable proprotein homodimer in its inactive (procytokine) state. The dashed line indicates the cleavable linker. Because the IL-2Rβγ binding site is "masked" in this format, the intact IgG-proIL-2 construct exhibits little or no IL-2 activity.

[0035] [Figure 3]Figure 3 shows the "activation" of IL-2 homodimers by protease cleavage of the cleavable linker in the TME. The protease-cleavable linker between IL-2 and IL-2Rα is stable in peripheral blood but can be cleaved by tumor proteases in the TME, thereby releasing active IL-2 at the tumor site. This may reduce toxicity in peripheral blood and increase antitumor activity.

[0036] [Figure 4] Figures 4A-4C show the ELISA binding activity of PD-1-proIL-2v to (A) human PD-1 or (B) Cyno PD-1 compared to PD-1 IgG, and (C) the binding activity of PD-L1-proIL-2v to PD-L1 compared to PD-L1 IgG.

[0037] [Figure 5] Figure 5 shows the results of a mixed lymphocyte reaction performed by co-culture of PBMC cells and allogeneic DCs with or without dose-escalating parental PD-1 antibodies or PD-1-proIL-2 added at the beginning of the assay. After 5 days, IFN-γ secretion in the culture supernatants was analyzed by ELISA.

[0038] [Figure 6] Figures 6A-6B show the proliferation of the human acute megakaryoblastic leukemia cell line M-07e induced by (A) MMP2 protease-activated PD-1-proIL-2v compared to human recombinant IL-2 and intact PD-1-proIL-2v, or (B) MMP2 protease-activated PD-L1-proIL-2v compared to human recombinant IL-2 and intact PD-L1-proIL-2v.

[0039] [Figure 7-1]Figures 7A-7D show the results of a STAT5 assay of intact and MMP2 protease-activated PD-1-proIL-2v on resting PBMCs from donor 1 (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D); legend in 7B above). [Figure 7-2] Same as above.

[0040] [Figure 8-1] Figures 8A-8D show the results of a STAT5 assay of intact and MMP2 protease-activated PD-L1-proIL-2v on resting PBMCs from donor 1 (CD4 T cells (A), CD8 T cells (B), regulatory T cells (C), and NK cells (D); legend in 8B above). [Figure 8-2] Same as above.

[0041] [Figure 9] 9A-9B show in vitro CD4 T cell activation and cytokine release. Dose-dependent (A) GM-CSF and (B) IFN-γ secretion by human polyclonal CD4 T cells upon 5 days of stimulation with increasing concentrations of either PD-1 antibody or intact or MMP2 protease-activated PD-1-proIL-2v is shown.

[0042] [Figure 10] Figure 10A shows the mean tumor volume measured over time in an A375-PBMC xenograft model, demonstrating the in vivo anti-tumor activity of PD-L1-proIL-2v with different linkers compared to anti-PD-L1 antibodies at the same molar concentration as single agents in inhibiting tumor growth. Figure 10B shows the mean tumor volume at day 24 for the same treatments as in 10A. Mice (n=5 or 6) were injected i.v. on days 0, 3, 7, 10, and 14; results are expressed as the mean ± SEM.

[0043] [Figure 11]Figure 11A shows the mean tumor volume measured over time in an HT-29-PBMC xenograft model, demonstrating the in vivo anti-tumor activity of PD-L1-proIL-2v with different linkers compared to anti-PD-L1 antibodies at the same molar concentration as single agents in inhibiting tumor growth. Figure 11B shows the mean tumor volume at day 23 for the same treatments as in 11A. Mice (n=6 for each group) were injected i.v. on days 0, 3, 7, and 10; results are expressed as the mean ± SEM.

[0044] [Figure 12] Figure 12A shows the mean tumor volume measured over time in an A375-PBMC xenograft model, demonstrating the in vivo antitumor activity of PD-1-proIL-2v with different linkers as single agents in inhibiting tumor growth. Figure 12B shows the mean tumor volume at day 29 for the same treatments as in 12A. Mice (n=5 or 6) were injected i.v. on days 0, 3, 7, and 10; results are expressed as mean±SEM.

[0045] [Figure 13] Figure 13A shows the mean tumor volume measured over time in an HT-29-PBMC xenograft model, demonstrating the in vivo anti-tumor activity of PD-1-proIL-2v with different linkers compared to anti-PD-1 antibodies as single agents in inhibiting tumor growth. Figure 13B shows the mean tumor volume at day 27 for the same treatments as in 13A. Mice (n=5 or 6) were injected i.v. on days 0, 4, 7, and 11; results are expressed as the mean ± SEM.

[0046] [Figure 14]Figure 14A shows the mean tumor volume measured over time in an A375-PBMC xenograft model, demonstrating the in vivo dose-dependent anti-tumor activity of PD-L1-proIL-2v and PD-1-proIL-2v as single agents in inhibiting tumor growth. Figure 14B shows the mean tumor volume at day 22 for the same treatments as in 14A. Mice (n=5 or 6) were injected i.v. on days 0, 3, 7, 10, and 14; results are expressed as the mean ± SEM.

[0047] [Figure 15] Figure 15A shows the mean tumor volume measured over time in an A375-PBMC xenograft model, demonstrating the in vivo antitumor activity of B7H3-proIL-2v and PD-1-proIL-2v as single agents in inhibiting tumor growth. Figure 15B shows the mean tumor volume on day 22 for the same treatments as in 15A. Mice (n=6) were injected i.v. on days 0 and 7; results are expressed as mean±SEM.

[0048] [Figure 16] Figure 16A shows the mean tumor volume measured over time in an A375-PBMC xenograft model, demonstrating the in vivo anti-tumor activity of PD-L1-proIL-2v and PD-L1-proIL-2wt as single agents in inhibiting tumor growth. Figure 16B shows the mean tumor volume at day 19 for the same treatments as in 16A. Mice (n=5 or 6) were injected i.v. on days 0, 3, and 7; results are expressed as the mean ± SEM.

[0049] [Figure 17]Figure 17A shows the mean tumor volume measured over time in an A375-PBMC xenograft model, demonstrating the in vivo anti-tumor activity of PD-1-proIL-2v and anti-PD-L1 antibodies, separately or in combination, in inhibiting tumor growth. Figure 17B shows the mean tumor volume at day 29 for the same treatments as in 17A. Mice (n=5 or 6) were injected i.v. on days 0, 3, 7, and 10; results are expressed as the mean ± SEM.

[0050] [Figure 18] Figure 18A shows the total drug (procytokines + activated cytokines) and procytokine concentrations over time of PD-1-proIL-2v in peripheral blood. Figure 18B shows the total drug and procytokine concentrations over time of PD-1-proIL-2v in tumors. Figure 18C shows the activated cytokine concentrations over time of PD-1-proIL-2v in tumors. Mice (n=3) were injected iv with P41222037 1 mg / kg on day 0; results are expressed as mean ± SEM.

[0051] [Figure 19] Figures 19A-19C show the number of CD3+ (A), CD4+ (B), and CD8+ (C) T cells in peripheral blood over time after treatment with PD-1-proIL-2v in a PBMC-humanized A375 xenograft model. Mice (n=5) were injected iv on days 0 and 7; results are expressed as mean ± SEM.

[0052] [Figure 20] Figures 20A-20C show the number of CD3+ (A), CD4+ (B), and CD8+ (C) T cells in tumors after treatment with PD-1-proIL-2v in an A375-PBMC xenograft model. Mice (n=3) were injected i.v. on days 0 and 8, and tumors were harvested on day 12; results are expressed as mean ± SEM.

[0053] [Figure 21]Figure 21A shows the concentration of masked or unmasked PD-1-proIL-2v over time in the peripheral blood of cynomolgus monkeys. Figure 21B shows the albumin levels over time in the peripheral blood of cynomolgus monkeys after treatment with masked or unmasked PD-1-proIL-2v. Monkeys (n=2) were injected iv with P41222037 or P41252037.

[0054] [Figure 22] Figures 22A-22C show the proliferation of the human acute megakaryoblastic leukemia cell line M-07e induced by human recombinant IL-2 and the protease-activated P41222037 (22A), P45412037 (22B), or P45422037 (22C) constructs compared to the intact proprotein (no protease activation) P41222037, P45412037, or P45422037 constructs, respectively.

[0055] [Figure 23-1] Figures 23A-23D show STAT5 phosphorylation in CD4 T cells, CD8 T cells, and Tregs upon treatment of resting PBMCs with both intact and MMP2-cleaved P41222037 (23A), P45412037 (23B), or P45422037 (23C) compared to human recombinant IL-2, as determined by flow cytometry. STAT5 phosphorylation analysis in CD4 T cells, CD8 T cells, and Tregs upon treatment of resting PBMCs with intact proprotein P41222037, P45412037, and P45422037 constructs is plotted together for comparison (23D). [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above.

[0056] [Figure 24-1]Figures 24A-24C show the effect of P78192037, P78202037, P78212037, P78222037, P78232037, and P78242037 (24A); P78252037, P78262037, P78272037, P78282037, P78292037, and P78302037 (24B); or P78312037, P78322037, P78332037, P78342037, P78352037, and P78362037 (24C) on the proliferation and proliferation of CD4 T cells, CD8 T cells, and CD8 T cells as determined by flow cytometry upon treatment of resting PBMCs with P78192037, P78202037, P78212037, P78222037, P78232037, and P78242037 (24A); P78252037, P78262037, P78272037, P78282037, P78292037, and P78302037 (24B); or P78312037, P78322037, P78332037, P78342037, P78352037, and P78362037 (24C) compared to human recombinant IL-2. STAT5 phosphorylation in T cells and Tregs is shown. [Figure 24-2] Same as above. [Figure 24-3] Same as above.

[0057] [Figure 25] FIG. 25 shows STAT5 phosphorylation in CD4 T cells, CD8 T cells and Tregs upon treatment of pre-activated PBMCs with the intact proprotein P41222037 construct as determined by flow cytometry.

[0058] [Figure 26] Figure 26 shows the amino acid sequences of the tested constructs at the junction of the fusion between the antibody heavy chain and the IL-2 variant. Exemplary junction sequences include various stable linker lengths, deletion of the C-terminal Lys of the heavy chain, and / or deletion of the N-terminal residue of IL-2. Sequence identifiers: LSPGK (SEQ ID NO: 249); LSPG (SEQ ID NO: 250); GGGS, GGGSGGGS (SEQ ID NO: 188); GGGSGGG (SEQ ID NO: 251); GGGSGG (SEQ ID NO: 252); GGGSG (SEQ ID NO: 253); APASSSTKK (SEQ ID NO: 254); PASSSTKK (SEQ ID NO: 255); ASSSTKK (SEQ ID NO: 256); SSSTKK (SEQ ID NO: 257); SSTKK (SEQ ID NO: 258); STKK (SEQ ID NO: 259); STLT (SEQ ID NO: 260). DETAILED DESCRIPTION OF THE INVENTION

[0059] Detailed Description Embodiments of the present disclosure relate to activatable proprotein homodimers comprising two separate but identical polypeptides, each polypeptide comprising, in N-terminal to C-terminal orientation, an antigen-binding fragment (Fab) region that specifically binds human PD-1 or human PD-L1, a hinge / Fc domain, a stable linker, an IL-2 protein, a protease-cleavable linker, and an IL-2Rα protein. In some examples, the homodimer is formed by the following binding interactions: binding of the hinge / Fc domain of one polypeptide to the hinge / Fc domain of the other polypeptide, and binding of each of the IL-2 proteins of one polypeptide to each of the IL-2Rα proteins of the other polypeptide. These binding interactions form a biologically inactive (proprotein) homodimer by masking the binding site of the IL-2 protein that would otherwise bind to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells. The proprotein homodimer remains inactive or substantially inactive in plasma.

[0060] In certain instances, the homodimer is targeted to the tumor microenvironment (TME) by the anti-PD-1 or anti-PD-L1 Fab and then activated within the TME by exposure to tumor-site proteases that cleave the protease-cleavable linker, thereby releasing the IL-2Rα protein and exposing the active site of the IL-2 protein. This allows for enhanced IL-2 signaling activity toward PD-1- or PD-L1-expressing cells due to a "cis-targeting" effect and / or synergy between the anti-tumor activity of the anti-PD-1 / anti-PD-L1 Fab and the immunostimulatory activity of the IL-2 protein.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in practicing or testing the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if each individual publication or reference was specifically and individually indicated to be incorporated herein by reference as if fully set forth. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety, in the manner described above for publications and references.

[0062] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0063] For purposes of this disclosure, the following terms are defined below.

[0064] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" includes "one element," "one or more elements," and / or "at least one element."

[0065] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0066] The terms "activatable proprotein," "proprotein," "activatable procytokine," "procytokine," "activatable prodrug," and "prodrug" are used interchangeably herein and refer to an activatable proprotein comprising at least a masking moiety and an active domain, or derivative / variant thereof, as described herein. In one embodiment, the proprotein may also comprise one or more protein domains.

[0067] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody, and can further be used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. As used herein, the term "antigen" includes a substance that, under appropriate conditions, is capable of inducing an immune response against that substance and reacting with the products of the immune response. In a broader sense, the term "antigen" includes any substance to which an antibody binds or against which an antibody is desired, regardless of whether the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods, independent of any immune response.

[0068] "Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, antagonists specifically bind to other agents or molecules. Full and partial antagonists are included.

[0069] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another drug or molecule. In some instances, agonists specifically bind to other drugs or molecules. Full and partial agonists are included.

[0070] As used herein, the term "amino acid" is intended to refer to both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis, as well as others, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, such as norleucine, norvaline, p-fluorophenylalanine, and ethionine, known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties of the reference amino acid, such as charge and charge spacing characteristics. For example, the organic structure that mimics arginine (Arg or R) has a positively charged portion that is located in a similar molecular space and has the same degree of flexibility as the e-amino group of the side chain of naturally occurring Arg amino acid.Mimetic also includes structures that are constrained to maintain optimal spacing and charge interaction of amino acid or amino acid functional groups.Those skilled in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0071] As used herein, a subject "at risk" of developing a disease or adverse reaction may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" indicates that the subject has one or more risk factors, which are measurable parameters that correlate with the development of disease, as described herein and known in the art. Subjects with one or more of these risk factors have a higher probability of developing a disease or adverse reaction than subjects without one or more of these risk factors(s).

[0072] "Biocompatible" refers to a material or compound that is generally not detrimental to the biological functions of a cell or subject and does not cause any degree of unacceptable toxicity, including allergic and disease states.

[0073] The term "bond" refers to a direct association between two molecules due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including, for example, interactions such as salt bridges and water bridges.

[0074] "Cis-targeting" refers to the preferential activity of one function from a multifunctional molecule (including bispecific or bifunctional molecules) on the same cells as a second function, e.g., binding activity, has an effect on. This approach offers the potential to optimize the use of functional modulators (inhibitors or stimulators of the function of a molecular target) to treat cancer and other diseases by limiting or enhancing their effects on specific cell types.

[0075] "Coding sequence" means any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

[0076] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.

[0077] "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action identified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they substantially affect the activity or action of the listed elements.

[0078] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers containing at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects on a subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, e.g., bacteria, typically gram-negative bacteria, although endotoxins can also be found in gram-positive bacteria, e.g., Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipo-oligosaccharides (LOS), which are found in the outer membrane of various gram-negative bacteria and represent a central pathogenic feature in the ability of these bacteria to cause disease. In humans, small amounts of endotoxin can cause fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0079] Therefore, in pharmaceutical production, since even small amounts can cause adverse effects in humans, it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers.Since temperatures above 300°C are typically required to decompose most endotoxins, depyrogenation ovens can be used for this purpose.For example, based on the main packaging material, such as a syringe or vial, a glass temperature of 250°C combined with a holding time of 30 minutes is often sufficient to achieve a 3-log reduction in endotoxin levels.Other methods for removing endotoxin are contemplated, including, for example, chromatography and filtration methods described herein and known in the art.

[0080] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus amebocyte lysate assay, which utilizes blood from horseshoe crabs, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, the endotoxin level can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1-10 EU.

[0081] The term "half-maximal effective concentration" or "EC 50 " refers to the concentration of an agent (e.g., an activatable proprotein) described herein that induces a response midway between baseline and the maximum after some specified exposure time; thus, the EC of a graded dose-response curve. 50indicates the concentration of the compound at which 50% of its maximal effect is observed. EC 50 The EC also indicates the plasma concentration required to obtain 50% of the maximum effect in vivo. 90 " refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. 90 " is "EC 50 and Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC 50 In some embodiments, the agent has an EC50 of about 1 nM or less. 50 It has a value.

[0082] "Immune response" refers to any immunological response originating from the immune system, including responses from cellular and humoral, innate and adaptive immune systems. Exemplary cellular immune cells include, for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and all subsets thereof. Cellular responses include, for example, effector function, cytokine release, phagocytosis, efferocytosis, translocation, trafficking, proliferation, differentiation, activation, suppression, cell-cell interaction, apoptosis, and the like. Humoral responses include, for example, IgG, IgM, IgA, and IgE responses and their corresponding effector functions.

[0083] The "half-life" of an agent, such as an activatable proprotein, can refer to the time it takes for the agent to lose half of its pharmacological, physiological, or other activity, compared to such activity at the time of administration into the serum or tissue of an organism, or compared to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of the agent to be reduced to half of the starting amount administered in the serum or tissue of an organism, compared to such amount or concentration at the time of administration into the serum or tissue of an organism, or compared to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0084] The terms "modulating" and "altering" include "increasing" or "enhancing" as well as "decreasing" or "reducing," typically by a statistically significant or physiologically significant amount or degree compared to a control. An "increased" or "enhanced" amount is typically a "statistically significant" amount, and is typically about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 500, 0 fold or more or at least about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 fold or more. An "increased" or "enhanced" amount includes an increase of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000% or more compared to a control. Amounts that are greater than or at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000% or more can also be included.A "decreased" or "reduced" amount is typically a "statistically significant" amount, and may be about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or even more than a control. or 5000 times lower or at most about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 times lower. A "decreased" or "reduced" amount can also include amounts that are 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, or 5000% less than a control. Examples of comparisons and "statistically significant" amounts are described herein.

[0085] The terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The term "enzyme" includes polypeptide or protein catalysts. These terms include modifications, such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The term "polypeptide" or "protein" refers to one or more chains of amino acids, each chain containing amino acids covalently linked by peptide bonds. The polypeptide or protein may include multiple chains having the sequence of a native protein, i.e., a protein occurring in nature and specifically produced by non-recombinant cells, or by genetically engineered or recombinant cells, linked together non-covalently and / or covalently by peptide bonds. The polypeptide or protein may include molecules having the amino acid sequence of a native protein or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules, typically composed of a heterologous polynucleotide sequence or combination of polynucleotide sequences not otherwise found in the cell.

[0086] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. The terms typically refer to polymeric forms of nucleotides, at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of any type of nucleotide. The terms include single- and double-stranded forms of DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules isolated free of total genomic DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but has been substantially isolated away from or purified free of total genomic DNA of the species from which the DNA segment is obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode a polypeptide are also included. Recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, and the like, are also included.

[0087] Additional coding or non-coding sequences may, but need not, be present within the polynucleotides described herein, and polynucleotides may, but need not, be linked to other molecules and / or support materials. Thus, polynucleotides or expressible polynucleotides, regardless of the length of the coding sequence itself, may be combined with other sequences, for example, expression control sequences.

[0088] The term "isolated" polypeptide or protein, as referred to herein, means that the subject protein (1) is free from at least some other proteins with which it is typically found in nature, (2) is essentially free from other proteins from the same source, e.g., from the same species, (3) is expressed by cells from a different species, (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) is not associated (by covalent or noncovalent interactions) with portions of proteins with which the "isolated protein" is naturally associated, (6) is operably associated (by covalent or noncovalent interactions) with polypeptides with which it is not naturally associated, or (7) is not naturally occurring. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0089] In certain embodiments, the "purity" of any given agent (e.g., an activatable proprotein) in a composition can be defined. For example, a particular composition can contain an agent, e.g., a polypeptide agent, that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or weight-by-weight basis, including, but by no means limited to, all decimal points and ranges therebetween, as measured, for example, by high-performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0090] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence to which another sequence is compared. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name and in tables and sequence listings.

[0091] Certain embodiments include biologically active "variants" and "fragments" of the proteins / polypeptides described herein, as well as the polynucleotides encoding them. "Variant" contains one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, for example, the Tables and Sequence Listing). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, similarity, or homology to a reference sequence described herein, and substantially retains the activity of the reference sequence. Also included are sequences that consist of a reference sequence or that differ from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acid or nucleotide additions, deletions, insertions, or substitutions, but that substantially retain at least one activity of the reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0092] The term "sequence identity" as used herein, or including, for example, "50% identical sequence to", refers to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleic acid bases (for example, A, T, C, G, I) or identical amino acid residues (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences over a comparison window can be performed by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA), or by visual inspection and selecting the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of the various methods. Reference may also be made to the BLAST family of programs, such as those disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0093] The term "solubility" refers to the ability of an agent (e.g., an activatable proprotein) provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration, either by mass of solute per unit volume of solvent (e.g., grams of solute per kg of solvent, grams per dL (100 mL), mg / mL, etc.), molality, molality, mole fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of that solute in that solvent under specified conditions, including temperature, pressure, pH, and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pHs, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain embodiments, solubility is measured in water or a physiological buffer, such as PBS or NaCl (with or without NaPO). In specific embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO). In certain embodiments, solubility is measured in a biological fluid (solvent), such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0094] A "subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes mammalian subjects, for example, human subjects.

[0095] "Substantially" or "essentially" means nearly wholly or completely, for example, 95%, 96%, 97%, 98%, 99% or more of some given amount.

[0096] " Statistically significant " means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include p-value, which is the frequency or probability that the observed event occurs when the null hypothesis is true. If the obtained p-value is less than significance level, the null hypothesis is rejected. In a simple example, significance level is defined as a p-value of 0.05 or less.

[0097] "Therapeutic response" refers to an improvement in symptoms (whether sustained or not) following administration of one or more therapeutic agents.

[0098] As used herein, the term "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" is the amount of agent (e.g., activatable proprotein, activated protein) required to elicit a desired biological response following administration.

[0099] As used herein, "treatment" of a subject (e.g., a mammal, e.g., a human) or cell is any type of intervention used in an attempt to alter the natural processes of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and can be performed either prophylactically or subsequent to the initiation of a pathological event or contact with an etiologic agent. Also included are "prophylactic" treatments, which may be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prophylaxis" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or its associated symptoms.

[0100] The term "wild-type" refers to the gene or gene product (eg, polypeptide) that is most frequently observed in a population, and is thus arbitrarily referred to as the "normal" or "wild-type" form of the gene.

[0101] Each embodiment in this specification applies to all other embodiments unless expressly stated otherwise. activatable proprotein homodimer

[0102] Certain embodiments include an activatable proprotein homodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, in N-terminal to C-terminal orientation, an antigen-binding fragment (Fab) region that specifically binds human PD-1 or human PD-L1, a hinge / Fc domain, a first linker, an IL-2 protein, a second linker, and an IL-2Rα protein, wherein the hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, the IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, and the IL-2Rα of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the binding is in vitro or in vivo to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell. The present invention relates to an activatable proprotein homodimer that masks the binding site of an IL-2 protein(s) to which it would otherwise bind in vivo, and wherein the second linker is a cleavable linker. In some embodiments, the total length of the stable linker provides for such binding and masking.

[0103] As noted above, the IL-2 protein(s) and IL-2Rα protein(s) interact or bind together, for example, via non-covalent interactions or certain covalent bonds (e.g., disulfide bonds). In some examples, the binding of the IL-2 protein(s) to the IL-2Rα protein(s) sterically blocks or impedes the binding of the IL-2 protein(s) to their cognate IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains expressed on immune cells. Exemplary IL-2 and IL-2Rα proteins are described elsewhere herein.

[0104] In some examples, the hinge / Fc domains of the first and second polypeptides dimerize together through at least one non-covalent interaction, at least one covalent bond (e.g., at least one disulfide bond), or any combination of non-covalent interactions and covalent bonds to further stabilize the activatable proprotein and / or further mask the binding of the IL-2 protein to its cognate receptor, e.g., IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains. Typically, however, the hinge / Fc domains of the first and second polypeptides do not bind together or dimerize through peptide or amide bonds. In some embodiments, the hinge / Fc domains bind together as homodimers, i.e., the homodimer is composed of two identical or nearly identical hinge / Fc domains. Thus, the hinge / Fc domains of the first and second polypeptides can be the same (or substantially the same) or different (e.g., knob-in-hole). Exemplary hinge / Fc domains are described herein.

[0105] As noted above, the second linker comprises a cleavable linker, e.g., a linker cleavable by a protease. In some examples, the first linker is a stable (e.g., physiologically stable) linker. In some examples, the protease is expressed in a target tissue or cell, e.g., a cancer tissue or cancer cell. Cleavage of the linker in this context releases the masking moiety, removing steric hindrance from the IL-2 protein and allowing selective activation of the IL-2 protein in diseased tissue or cells (e.g., TME) compared to normal or healthy tissue or cells. Such selective and localized activation not only reduces wasteful consumption of administered IL-2, thereby increasing its half-life, but also enhances tissue penetration of IL-2 and reduces its undesirable systemic effects, among other advantages. Exemplary linkers are described herein.

[0106] In some embodiments, the homodimeric binding between the first and second polypeptides allosterically inhibits the binding of IL-2 proteins to their targets, e.g., cognate IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains on the surface of immune cells. In these and related embodiments, the IL-2 portion of the activatable proprotein can be allosterically inhibited, if desired, in vivo or using target displacement techniques available in the art. or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer, as appropriate, as compared to the binding of the active domain or IL-2 protein alone, as measured in an in vitro assay.

[0107] In certain instances, the anti-PD-1 or anti-PD-L1 Fab not only improves targeting of the activatable IL-2 proprotein (or procytokine) module to the TME, but also provides increased anti-cancer / immunostimulatory activity in addition to that of the IL-2 protein when the latter is activated by protease cleavage of the second linker.

[0108] In a specific embodiment, the first and second polypeptides of the activatable proprotein homodimer include active or otherwise functional fragments and variants of the following sequences: human IgG1 CH1 and CL domains (kappa), an IgG1 hinge (SEQ ID NO: 42), a modified IgG1 Fc domain with LALA and P329A mutations (see the CH2 domain of SEQ ID NO: 57), an IgG1 CH3 domain (see the CH3 domain of SEQ ID NO: 58), a stable linker (e.g., 4 amino acids in length, e.g., GGGS; SEQ ID NO: 188), an IL-2 protein (SEQ ID NO: 84 or 85, optionally with R38D, K43E, C125S mutations), a protease-cleavable linker (e.g., PLGLAGSGRSDNQGA; SEQ ID NO: 93), and an IL-2Rα protein (SEQ ID NO: 88 or 90, optionally with D6R and E29K mutations), as described herein. Fab (SEQ ID NOs: 25 and 26).

[0109] In a specific embodiment, the first and second polypeptides of the activatable proprotein homodimer comprise SEQ ID NO: 146 (anti-PD-1 heavy chain, in combination with a modified IgG1 Fc domain, a stable linker, IL-2, a protease-cleavable linker, IL-2Rα) and SEQ ID NO: 147 (anti-PD-1 light chain).

[0110] The individual components of exemplary activatable proproteins are described in more detail herein.

[0111] Anti-PD-1 and Anti-PD-L1 and Anti-B7H3 Fab Regions. The activatable proproteins described herein comprise at least one antigen-binding fragment (Fab) region that specifically binds to human PD-1, human PD-L1, or human B7H3. The "Fab" region is composed of one constant domain and one variable domain from each of the heavy and light chains of an immunoglobulin molecule, e.g., a VL / CL region or domain connected to a VH / CH1 region, or a domain fused at its C-terminus to an Fc domain, optionally via a linker or hinge (hinge / Fc domain). The VL:VH and CL:CH1 regions of the Fab are typically linked together as a covalent heterodimer. In certain embodiments, the CL domain is a kappa chain. In some embodiments, the CL domain is a lambda chain. In some embodiments, the CH1 domain is an IgA, IgD, IgE, IgG, or IgM domain, e.g., an IgA1, IgA2, IgG1, IgG2, IgG2, IgG3, or IgG4 CH1 domain. In a specific embodiment, the CL domain is a kappa chain and the CH1 domain is an IgG1 domain.

[0112] In certain embodiments, the antibodies or Fab regions described herein comprise a set of heavy and light chain CDRs sandwiched between a set of heavy and light chain framework regions (FRs), which provide support for the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are referred to as "CDR1," "CDR2," and "CDR3," respectively. Thus, the antigen-binding site comprises six CDRs (VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3) comprising the CDR sets from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of several antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0113] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact a bound antigen; however, FRs, particularly those directly adjacent to the CDRs, are primarily responsible for folding the V region into the antigen-binding site. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are conserved structural regions of FRs that affect the folded shape of the CDR loops into a particular "canonical" structure. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between antibody heavy and light chains.

[0114] In certain embodiments, the Fab region is humanized. These embodiments refer to chimeric molecules, typically prepared using recombinant techniques, that have an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site can comprise either a complete variable domain fused onto a constant domain, or only CDRs grafted onto appropriate framework regions in the variable domain. The epitope-binding site can be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but leaves open the possibility of an immune response to the foreign variable region (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Exemplary methods for antibody humanization include those described in US Pat. No. 7,462,697.

[0115] Another approach focuses not only on providing human-derived constant regions, but also on modifying variable regions and reshaping them to be as close as possible to human form.It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs), which respond differently to the epitope in question and determine binding ability, and four framework regions (FRs) adjacent to them that are relatively conserved in a given species and presumably provide a scaffold for the CDRs.When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting the CDRs derived from the non-human antibody onto the FRs present in the modified human antibody. Application of this approach to various antibodies has been reviewed by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio / Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody or Fab region preserves all CDR sequences (e.g., a humanized mouse antibody or Fab containing all six CDRs from the mouse antibodies).In certain embodiments, a humanized antibody or Fab region has one or more (one, two, three, four, five, six) CDRs that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0116] The binding properties of antibodies and Fab regions can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the antibody or Fab region has a binding affinity of about ≦10 -7 M~about 10 -8 M or about ≦10 -7 M~about 10 -8 specifically binds to a target molecule, e.g., PD-1 or PD-L1 protein, or an epitope or complex thereof, with an equilibrium dissociation constant in the range of M. In some embodiments, the equilibrium dissociation constant is about ≦10 -9 M~approx.≦10 -10 M or about ≦10 -9 M~approx.≦10 -10In certain exemplary embodiments, the antibody or antigen-binding fragment thereof has a mAb content of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM. an affinity (Kd or EC ) for the PD-1 or PD-L1 protein (to which it specifically binds) of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM 50 )

[0117] A molecule such as an antibody or Fab region is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell, substance, or particular epitope more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it reacts or associates with alternative cells, substances, or epitopes. An antibody "specifically binds" or "preferentially binds" to a target molecule or epitope if it binds, for example, by a statistically significant amount, with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances or epitopes. Typically, one member of a pair of molecules exhibiting specific binding has an area or cavity on its surface that specifically binds to, and is therefore complementary to, a particular spatial and / or polar organization of the other member of the pair. Thus, the members of the pair have the property of specifically binding to each other. For example, an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds to that specific epitope with higher affinity, avidity, more easily, and / or with longer duration than when it binds to other epitopes.By reading this definition, it is also understood that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.This term is also applicable, for example, when an antibody is specific to a particular epitope carried by several antigens, and in this case, a specific binding member that carries an antigen-binding fragment or domain can bind to various antigens that carry that epitope; for example, it can be cross-reactive with several different forms of target antigens from multiple species that share a common epitope.

[0118] Immunological binding generally refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and the antigen for which it is specific, for example, by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and / or hydrophobic attraction or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd indicating a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculating the concentrations and the actual rates of association and dissociation. The ratio Koff / Kon allows for the cancellation of all parameters not related to affinity and is therefore equal to the dissociation constant Kd. As used herein, the term "affinity" includes the equilibrium constant for the reversible binding of two drugs, and is expressed as Kd or EC 50 The affinity of an antibody for a PD-1 or PD-L1 protein or epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution.

[0119] Programmed cell death protein 1 (PD-1; CD279 (cluster of differentiation 279)) refers to a protein expressed on the surface of cells that regulates the immune response to cells of the human body, for example, by downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity (see Uniprot:Q15116). PD-1 is an immune checkpoint that promotes apoptosis of antigen-specific T cells in lymph nodes and reduces apoptosis in regulatory T cells (anti-inflammatory, suppressor T cells). Thus, in certain embodiments, the Fab region specifically binds to the human PD-1 protein sequence set forth in Uniprot:Q15116. Anti-PD-1 antibodies are known in the art (see, e.g., U.S. Patent Nos. 8,008,449; 8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342; 9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Patent Application Nos. 2012 / 0039906; 2015 / 0203579). For example, in specific embodiments, the Fab region is derived from an anti-PD-1 antibody selected from nivolumab, pembrolizumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, MGA012, AMP-22, and AMP-514.

[0120] Programmed death-ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that binds to its receptor PD-1, expressed on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition (see Uniprot: Q9NZQ7). In certain embodiments, the Fab region specifically binds to the human PD-L1 protein sequence set forth in Uniprot: Q9NZQ7. Anti-PD-L1 antibodies are known in the art (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; and 9,439,962). For example, in a specific embodiment, the Fab region is derived from an anti-PD-L1 antibody selected from atezolizumab, avelumab, and durvalumab.

[0121] In certain embodiments, an anti-PD-1 or anti-PD-L1 Fab region is characterized by or comprises a heavy chain variable (VH) region sequence comprising complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and a light chain variable (VL) region sequence comprising complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3 sequences. Exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2, and VLCDR3 sequences are provided in Tables P1 and P2 below. [Table P1-1] [Table P1-2] [Table P2-1] [Table P2-2]

[0122] Thus, in certain embodiments, the anti-PD-1 Fab region comprises a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions (underlined) from Table P1, and a corresponding VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions (underlined) from Table P1. Also included are variants thereof that bind to human PD-1, e.g., variants having a total of one, two, three, four, five, or six alterations in the combined CDR regions, e.g., the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3 sequences described herein. Exemplary "alterations" include amino acid substitutions, additions, and deletions. In certain embodiments, the anti-PD-1 Fab region comprises a VH region from Table P1 and a corresponding VL region from Table P1. In certain embodiments, the VH region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table P1, and the VL region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding sequence selected from Table P1. Also included are variants thereof that bind to human PD-1, e.g., variants with one, two, three, four, five, or six alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0123] In certain embodiments, the anti-PD-L1 Fab region comprises a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions (underlined) from Table P2, and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions (underlined) from Table P2. Variants thereof that bind to human PD-L1 are also included, such as variants with a total of one, two, three, four, five, or six alterations in the combined CDR regions, e.g., the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3 sequences described herein. Exemplary "alterations" include amino acid substitutions, additions, and deletions. In certain embodiments, the anti-PD-L1 Fab region comprises a VH region from Table P2 and the corresponding VL region from Table P2. In certain embodiments, the VH region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from Table P2, and the VL region comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding sequence selected from Table P2. Also included are variants thereof that bind to human PD-L1, for example, variants with one, two, three, four, five, six alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0124] CD276 (B7H3) is an immune checkpoint molecule involved in regulating T cell-mediated immune responses and expressed on some solid tumors. It plays a protective role in tumor cells, for example, by inhibiting natural killer-mediated cytolysis and potentially other anti-tumor immune responses. In certain embodiments, B7H3 is human B7H3, or a domain thereof. In certain embodiments, the anti-B7H3 Fab region specifically binds to a human B7H3 protein, e.g., a domain of human B7H3 selected from one or more of the Ig-like V-1 type domain, the Ig-like C2-1 type domain, the Ig-like V-2 type domain, and the Ig-like C2-2 type domain. In a specific embodiment, the Fab region has a K of about 0.4 or 0.5 nM (400 or 500 pM) or lower. D and specifically binds to human BH73.

[0125] In certain embodiments, the anti-B7H3 Fab region is characterized by or comprises a VH sequence comprising complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and a VL sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences. Exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2, and VLCDR3 sequences are provided in Table P3 below. [Table P3-1] [Table P3-2]

[0126] Thus, in certain embodiments, the anti-B7H3 Fab region comprises a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions (underlined) from Table P3, and a corresponding VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions (underlined) from Table P3. Also included are variants thereof that bind to human B7H3, e.g., variants having a total of one, two, three, four, five, or six alterations in the combined CDR regions, e.g., the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3 sequences described herein. Exemplary "alterations" include amino acid substitutions, additions, and deletions. In certain embodiments, the anti-B7H3 Fab region comprises a VH region from Table P3 and a corresponding VL region from Table P3. In certain embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from Table P3, and the VL region comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding sequence selected from Table P3. Also included are variants thereof that bind to human B7H3, e.g., variants having one, two, three, four, five, or six alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0127] Antibodies or Fab regions can be prepared by any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific to a polypeptide of interest can be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, or improvements thereto. Methods using transgenic animals such as mice to express human antibodies or Fab regions are also included. See, for example, Neuberger et al., Nature Biotechnology 14:826, 1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. The structure and location of immunoglobulin variable domains can be determined with reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987 and its revised editions.

[0128] It will be understood that any one or more of the above-described anti-PD-1 Fabs or anti-PD-L1 Fabs or anti-B7H3 Fabs can be combined with any of the other components described herein, such as the CH1 domains, hinge / Fc domains, IL-2 protein, IL-2Rα protein, and linkers described herein, to generate one or more activatable proproteins.

[0129] Hinge / Fc domain. Certain activatable proprotein homodimers contain a hinge / Fc domain. The hinge region (found in IgG, IgA, and IgD) acts as a flexible spacer, allowing the Fab portion to move freely relative to the Fc domain. In contrast to the constant region, the hinge region is structurally diverse, varying in both sequence and length between immunoglobulin classes and subclasses. The hinge region may also contain one or more glycosylation sites, including several structurally distinct types of sites for carbohydrate binding. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, conferring significant resistance of the hinge region polypeptide to intestinal proteases. Residues in the hinge-proximal region of the CH2 domain can also influence the specificity of the interaction between an immunoglobulin and its respective Fc receptor(s) (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0130] The term "Fc domain" or "Fc fragment" or "Fc" refers to a protein containing one or more CH2, CH3, and / or CH4 domains from one or more selected immunoglobulins, including fragments, variants, and combinations thereof. An "Fc domain" may also include one or more hinge regions of the heavy chain constant region of an immunoglobulin. In certain embodiments, the Fc domain does not contain one or more of the CH1, CL, VL, and / or VH regions of an immunoglobulin.

[0131] The Fc domain may be derived from the CH2 domain, CH3 domain, CH4 domain, and / or hinge region(s) of any one or more immunoglobulin classes, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, including subclasses and combinations thereof. In some embodiments, the Fc domain is derived from an IgA immunoglobulin, including subclasses IgA1 and / or IgA2. In certain embodiments, the Fc domain is derived from an IgD immunoglobulin. In certain embodiments, the Fc domain is derived from an IgE immunoglobulin. In some embodiments, the Fc domain is derived from an IgG immunoglobulin, including subclasses IgG1, IgG2, IgG2, IgG3, and / or IgG4. In certain embodiments, the Fc domain is derived from an IgM immunoglobulin. Exemplary hinge and Fc domain sequences are provided in Table F1 below. [Table F1-1] [Table F1-2]

[0132] Thus, in some embodiments, the hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a hinge sequence selected from Table F1, e.g., an IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 hinge region selected from Table F1. In certain embodiments, the Fc domain comprises a sequence selected from Table F1, e.g., an IgA1 CH2, CH3 or combined CH2CH3 sequence from Table F1, an IgA2 CH2, CH3 or combined CH2CH3 sequence, an IgD CH2, CH3 or combined CH2CH3 sequence, an IgE CH2, CH3, CH4 or combined CH2CH3 or CH2CH3CH4 sequence, an IgG1 CH2, CH3 or combined CH2CH3 or CH2CH3CH4 sequence. In certain embodiments, the hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IgG2 CH2, CH3, or combined CH2CH3 sequence of IgG2, a CH2, CH3, or combined CH2CH3 sequence of IgG3, a CH2, CH3, or combined CH2CH3 sequence of IgG4, or a CH2, CH3, CH4, or combined CH2CH3 or CH2CH3CH4 sequence of IgM. In certain embodiments, the hinge is of the same Ig class as the Fc domain.

[0133] In certain embodiments, the Fc domain is a modified Fc domain. Such modifications can affect, among other properties described herein, the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C max , t max , C min, fluctuation), its immunogenicity, its complement fixation or activation, and / or CDC / ADCC / ADCP-related activity of the Fc region compared to the corresponding wild-type Fc sequence.

[0134] In some embodiments, the modified Fc domain does not bind to or does not substantially bind to FcγR. Examples of FcγR include FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. FcγRI (CD64) is expressed on macrophages and dendritic cells and plays a role in phagocytosis, respiratory burst, cytokine stimulation, and dendritic cell endocytic transport. Expression of FcγRI is upregulated by both GM-CSF and gamma-interferon (gamma-IFN) and downregulated by interleukin-4 (IL-4). FcγRIIa is expressed on polymorphonuclear leukocytes (PMN), macrophages, dendritic cells, and mast cells. FcγRIIa plays a role in phagocytosis, respiratory burst, and cytokine stimulation. FcγRIIa expression is upregulated by GM-CSF and gamma-IFN and reduced by IL-4. FcγIIb is expressed on B cells, PMNs, macrophages, and mast cells. FcγIIb inhibits immunoreceptor tyrosine-based activation motif (ITAM)-mediated responses and is therefore an inhibitory receptor. FcγRIIc expression is upregulated by intravenous immunoglobulin (IVIG) and IL-4 and reduced by gamma-IFN. FcγRIIc is expressed on NK cells. FcγRIIIa is expressed on natural killer (NK) cells, macrophages, mast cells, and platelets. This receptor is involved in phagocytosis, respiratory burst, cytokine stimulation, platelet aggregation and degranulation, and NK-mediated ADCC. FcγRIII expression is upregulated by C5a, TGF-β, and gamma-IFN and downregulated by IL-4. FcγRIIIb is a GPI-linked receptor expressed on PMNs.

[0135] In a specific embodiment, the modified Fc domain comprises a L234A / L235A ("LALA") mutation and / or a P329A or P329G mutation (EU numbering) (see, e.g., the CH2 domain of SEQ ID NO: 57). In certain embodiments, the Fc domain or modified Fc domain retains normal (wild-type) or substantially normal binding to the neonatal Fc receptor (FcRn). In a specific embodiment, the Fc region comprises an IgG1 hinge region of SEQ ID NO: 42, a modified IgG1 CH2 domain of SEQ ID NO: 57, and an IgG1 CH3 domain of SEQ ID NO: 58, including functional or active fragments and variants thereof, as described herein.

[0136] It will be understood that any one or more of the hinge and Fc domains described above may be combined with any of the other components described herein, for example, the anti-PD-1 Fabs, anti-PD-L1 Fabs, IL-2 proteins, IL-2Rα proteins, and linkers described herein, to generate one or more activatable proproteins.

[0137] IL-2 Protein. The activatable proproteins described herein contain at least one "IL-2 protein" (or interleukin-2 protein), including human IL-2 protein. IL-2 is a cytokine that signals through the IL-2 receptor (IL-2R), a complex composed of up to three chains called the α (CD25), β (CD122), and γc (CD132) chains. IL-2 is produced by T cells in response to antigenic or mitogenic stimuli and is required for T cell proliferation and other activities important for regulating the immune response. IL-2 can stimulate B cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells, among other immune cells.

[0138] IL-2 is a 15-16 kDa protein composed of a signal peptide (residues 1-20) and the active mature protein (residues 21-153). An exemplary human IL-2 amino acid sequence is provided in Table S1 below. [Table S1-1] [Table S1-2] [Table S1-3]

[0139] Thus, in certain embodiments, an IL-2 protein comprises, consists of, or consists essentially of an amino acid sequence selected from Table S1, or an active variant or fragment thereof that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S1. In some embodiments, an "active" IL-2 protein or fragment or variant is characterized by its ability to bind, for example, in vitro or in vivo, to the IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains present on the surface of immune cells, and its ability to stimulate downstream signaling activity in the absence of steric hindrance by a masking moiety as described herein. Examples of downstream signaling activity include IL-2-mediated signaling through one or more of the JAK-STAT, PI3K / Akt / mTOR, and MAPK / ERK pathways, including combinations thereof. Together, IL-2 signaling stimulates an array of downstream pathways that result in responses that have important roles in the development, function, and survival of CD4 T cells, CD8 T cells, NK cells, NKT cells, macrophages, and intestinal intraepithelial lymphocytes, among others.

[0140] In certain embodiments, the IL-2 protein is the mature form of IL-2, or an active variant or fragment thereof comprising, consisting of, or consisting essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to amino acids 21-153 or 26-153 of SEQ ID NO: 68. In some embodiments, the IL-2 protein comprises a C145X substitution as defined by SEQ ID NO: 68, where X is any amino acid. In a specific embodiment, the IL-2 protein comprises a C145S substitution as defined by SEQ ID NO: 68.

[0141] Certain IL-2 proteins comprise, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 69 (mature human IL-2 with a C125S substitution). In some embodiments, an active variant or fragment of SEQ ID NO: 69 retains the S125 residue as defined herein.

[0142] Certain IL-2 proteins contain one or more defined amino acid substitutions compared to the exemplary amino acid sequences in Table S1. For example, some IL-2 proteins contain one or more amino acid substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C as defined by SEQ ID NO: 69. In some embodiments, the IL-2 protein forms a disulfide bond with an IL-2 binding protein (e.g., IL-2Ra) through one or more cysteine ​​substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C. Certain IL-2 proteins contain one or more amino acid substitutions at positions 69, 74, and / or 128 as defined by SEQ ID NO: 69, including combinations thereof, as well as when, for example, one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO: 69. Some IL-2 proteins include, but are not limited to, one or more amino acid substitutions, including combinations thereof, such as R38A, R38D, and R38K; F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, and F42I; K43E; Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, Y45R ... and one or more amino acid substitutions at positions R38, F42, K43, Y45, E62, E68 and / or L72 as defined by SEQ ID NO: 69, including those selected from: 45R and Y45K; E62A and E62L; E68A and E68V; and L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K.Specific examples include when an IL-2 protein contains one or a combination of amino acid substitutions selected from: F42A, Y45A, and L72G; R38K, F42Q, Y45N, E62L, and E68V; R38K, F42Q, Y45E, and E68V; R38A, F42I, Y45N, E62L, and E68V; R38K, F42K, Y45R, E62L, and E68V; R38K, F42I, Y45E, and E68V; and R38A, F42A, Y45A, and E62A. Some IL-2 proteins contain one or a combination of amino acid substitutions in T3 and / or E61 as defined by SEQ ID NO: 69, e.g., T3A and / or E61S. Certain IL-2 proteins (e.g., mature IL-12 or residues 26-153) contain a combination of R38D and K43E (see, e.g., SEQ ID NO: 85). Thus, an IL-2 protein can contain any one or more of the amino acid substitutions described above, including combinations thereof.

[0143] It will be understood that any one or more of the IL-2 proteins described above can be combined with any of the other components described herein, such as the anti-PD-1 Fab, anti-PD-L1 Fab, hinge / Fc domain, IL-2Rα protein, and linker described herein, to generate one or more activatable proproteins.

[0144] IL-2Rα Protein. The activatable proproteins described herein contain at least one "IL-2Rα protein" (or interleukin-2 receptor-α protein), including human IL-2Rα protein. The IL-2 receptor is composed of three subunits: IL-2Rα, CD122, and CD132. IL-2Rα specifically binds IL-2 with very high affinity and is capable of binding IL-2 independently of other subunits. Exemplary IL-2Rα protein sequences are provided in Table S2. [Table S2-1] [Table S2-2]

[0145] Thus, in certain embodiments, the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence selected from Table S2, or an active variant or fragment thereof that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S2 and that binds to IL-2 protein. In some embodiments, the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to amino acids 22-187 or 22-240 of SEQ ID NO: 86 (full-length wild-type human IL-2Rα).

[0146] Certain IL-2Rα proteins contain one or more defined amino acid substitutions compared to the exemplary amino acid sequences in Table S2. For example, in some examples, the IL-2Rα protein contains one or more amino acid substitutions in D6 and / or E29, e.g., D6R and / or E29K, as defined by SEQ ID NO: 87 or 88. In some examples, the IL-2Rα protein contains one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C as defined by SEQ ID NO: 88 (the Sushi1-Sushi2 domains of human IL-2Rα). In some examples, the IL-2Rα protein contains an alanine substitution at position 49 and / or 68 as defined by SEQ ID NO: 88. In some embodiments, the IL-2Rα protein contains a K38S substitution as defined by SEQ ID NO: 88. Thus, the IL-2Rα protein can contain any one or more of the amino acid substitutions described above, including combinations thereof. In specific embodiments, the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% similar to SEQ ID NO: 90, including when the IL-2Rα protein retains a D6R and / or E29K substitution.

[0147] In certain of these and related embodiments, the IL-2Rα protein forms at least one disulfide bond with the IL-2 protein via one or more of the above-mentioned cysteines and one or more cysteines in the IL-2 protein. In specific embodiments, the IL-2Rα and IL-2 protein form at least one disulfide bond between one or more pairs of cysteines selected from IL2-K35C and IL2Rα-D4C, IL2-R38C and IL2Rα-D6C, IL2-R38C and IL2Rα-H120C, IL2-T41C and IL2Rα-I118C, IL2-F42C and IL2Rα-N27C, IL2-E61C and IL2Rα-K38C, IL2-E61C and IL2Rα-S39C, and IL2-V69C and IL2Rα-L42C. In certain embodiments, as noted above, the bond (e.g., disulfide bond) between the IL-2 protein and the IL-2Rα protein is reg This masks or sterically hindrances the binding site of the IL-2 protein, which preferentially binds to the IL-2Rα / β / γc chain expressed on the IL-2Rα / β / γc receptor.

[0148] It will be understood that any one or more of the IL-2Rα proteins described above can be combined with any of the other components described herein, such as the anti-PD-1 Fab, anti-PD-L1 Fab, hinge / Fc domain, IL-2 protein, and linker described herein, to generate one or more activatable proproteins.

[0149] Linker. As noted above, in certain embodiments, each polypeptide comprises at least a first linker and a second linker, typically a peptide linker. In some embodiments, the first linker is a non-cleavable linker, i.e., a physiologically stable linker. In some embodiments, the second linker is a cleavable linker, for example, a cleavable linker comprising a protease cleavage site.

[0150] In some embodiments, the first linker and / or the second linker are about 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3 amino acids in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acids in length. In certain embodiments, the first linker is a cleavable linker and the second linker is a non-cleavable linker. In some embodiments, the first linker is a non-cleavable linker and the second linker is a cleavable linker, hi some embodiments, both linkers are cleavable linkers.

[0151] In some embodiments, the cleavable linker comprises at least one protease cleavage site.Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., J. Gener. Virol. 78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589-594, 2004).In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of metalloproteases, serine proteases, cysteine ​​proteases, and aspartic acid proteases. In certain embodiments, the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A, and cathepsin B.

[0152] Exemplary cleavable linker sequences are provided in Table S3. [Table S3-1] [Table S3-2]

[0153] Thus, in certain embodiments, the cleavable linker is selected from Table S3. Additional examples of cleavable linkers include amino acid sequences that are cleaved by serine proteases, such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Illustrative examples of thrombin-cleavable amino acid sequences include, but are not limited to, -Gly-Arg-Gly-Asp-(SEQ ID NO: 160), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO: 161), -Gly-Arg-Gly-Asp-Ser-(SEQ ID NO: 162), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO: 163), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Illustrative examples of elastase-cleavable amino acid sequences include, but are not limited to, -Ala-Ala-Ala-, -Ala-Ala-Pro-Val- (SEQ ID NO: 164), -Ala-Ala-Pro-Leu- (SEQ ID NO: 165), -Ala-Ala-Pro-Phe- (SEQ ID NO: 166), -Ala-Ala-Pro-Ala- (SEQ ID NO: 167), and -Ala-Tyr-Leu-Val- (SEQ ID NO: 168).

[0154] Cleavable linkers also include amino acid sequences that can be cleaved by matrix metalloproteinases, such as collagenases, stromelysins, and gelatinases. Illustrative examples of matrix metalloproteinase-cleavable amino acid sequences include, but are not limited to, -Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO: 169), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO: 170), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 171), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO: 172), where Y and Z are amino acids. Illustrative examples of collagenase-cleavable amino acid sequences include, but are not limited to, -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO: 173), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO: 174), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO: 175), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO: 176), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO: 177), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO: 178), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO: 179), wherein Z is an amino acid. An illustrative example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 180); an example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 181).

[0155] Cleavable linkers also include amino acid sequences that can be cleaved by angiotensin-converting enzyme, such as -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO: 182), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 183). Cleavable linkers also include amino acid sequences that can be degraded by cathepsin B, such as Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO: 184), Gly-Phe-Leu-Gly- (SEQ ID NO: 185), and Phe-Lys.

[0156] In certain embodiments, the cleavable linker has a half-life at pH 7.4, 25°C, e.g., physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue), of about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours, or less than about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours, or any half-life in between.

[0157] Typically, at least one of the first or second linker is a non-cleavable linker.Exemplary non-cleavable linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.Particular non-cleavable linker sequences contain Gly, Ser and / or Asn residues.Other near-neutral amino acids, such as Thr and Ala, can also be used in peptide linker sequences as desired.

[0158] Certain exemplary non-cleavable linkers include Gly, Ser and / or Asn-containing linkers, such as: [G] x , [S] x , [N] x , [GS]x , [GGS] x , [GSS] x , [GSGS] x (SEQ ID NO: 186), [GGSG] x (SEQ ID NO: 187), [GGGS] x (SEQ ID NO: 188), [GGGGS] x (SEQ ID NO: 189), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO: 190), [GGNG] x (SEQ ID NO: 191), [GGGN] x (SEQ ID NO: 192), [GGGGN] x (SEQ ID NO: 193) linker, wherein: x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more. Other combinations of these and related amino acids will be apparent to those skilled in the art.

[0159] Additional examples of non-cleavable linkers include the following amino acid sequences: Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 194); Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 195); Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys-(SEQ ID NO: 197); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO: 198).

[0160] Further non-limiting examples of non-cleavable linkers include: DGGGS (SEQ ID NO: 199); TGEKP (SEQ ID NO: 200) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO: 201) (Pomerantz et al. 1995); (GGGGS) n(SEQ ID NO: 202) (Kim et al., PNAS. 93:1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 203) (Chaudhary et al., PNAS. 87:1066-1070, 1990); KESGSVGSSEQLAQFRSLD (SEQ ID NO: 204) (Bird et al., Science. 242:423-426, 1988), GGRRGGGS (SEQ ID NO: 205); LQRDGERP (SEQ ID NO: 206); LRQKDGGGSERP (SEQ ID NO: 207); LRQKd(GGGS)2ERP (SEQ ID NO: 208). In a specific embodiment, the linker comprises a Gly3 linker sequence containing three glycine residues. In certain embodiments, flexible linkers can be rationally designed using computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994), or by phage display methods.

[0161] In some embodiments, the linker comprises a spacer element and a cleavable element to make the cleavable element more accessible to the enzyme responsible for cleavage.

[0162] It will be understood that any one or more of the above-described linkers may be combined with any one or more of the anti-PD-1 Fab, anti-PD-L1 Fab, hinge / Fc domain, IL-2 protein, and IL-2Rα protein described herein to form an activatable proprotein homodimer.

[0163] Exemplary activatable proprotein homodimers are provided in Table S4. [Table S4-1] [Table S4-2] [Table S4-3] [Table S4-4] [Table S4-5] [Table S4-6] [Table S4-7] [Table S4-8] [Table S4-9] [Table S4-10] [Table S4-11]

[0164] Thus, in certain embodiments, the activatable proprotein comprises first and second polypeptides comprising, consisting of, or consisting essentially of amino acid sequences at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S4 (i.e., chains 1 and 2), and VL / CL region polypeptides at least 80, 85, 90, 95, 98, or 100% identical to the corresponding sequences from Table S4 (i.e., chains 3 and 4).

[0165] In certain additional embodiments, the first polypeptide is distinct from the second polypeptide and forms a heterodimeric proprotein. For example, in some embodiments, the Fc domain from the first polypeptide and the Fc domain from the second polypeptide are modified (e.g., using knob-and-hole mutations) to promote Fc heterodimerization. In some embodiments, the IL-2 protein of the first polypeptide and the IL-2Rα protein of the second polypeptide are modified (mutated) to form a specific or preferential binding pair, and / or the IL-2 protein of the second polypeptide and the IL-2Rα protein of the first polypeptide are modified (mutated) to form a specific or preferential binding pair. In certain of these and related embodiments, the modifications to the IL-2 and IL-2Rα proteins prevent homodimer formation between (i) the IL-2 / IL-2Rα of the first polypeptide and (ii) the IL-2 / IL-2Rα protein of the second polypeptide. Methods of Use and Pharmaceutical Compositions

[0166] Certain embodiments include methods of treating a disease or condition, ameliorating the symptoms of a disease or condition, and / or reducing the progression of a disease or condition in a subject in need thereof, comprising administering to the subject at least one activatable proprotein described herein. Also included are methods of enhancing an immune response in a subject, comprising administering to the subject at least one activatable proprotein described herein. In certain embodiments, the disease is cancer. In some embodiments, the cancer expresses or overexpresses PD-L1.

[0167] In some embodiments, after administration, the activatable proprotein is activated in cells or tissues via protease cleavage, exposing an IL-2 protein binding site that binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo, thereby generating an activated protein. In certain embodiments, protease cleavage occurs in cancer cells or cancer tissues. Typically, the activated protein has at least one immunostimulatory IL-2 activity, for example, by binding to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vivo, thereby stimulating the immune cells. In certain embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0168] In some embodiments, administration and activation of the activatable proprotein to produce an activated protein increases an anti-cancer immune response in a subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to a control. In some embodiments, administration and activation of an activatable proprotein to produce an activated protein increases cancer cell killing in a subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to a control.

[0169] In certain embodiments, the activating anti-PD-1 Fab / IL-2 protein stimulates an increased (e.g., synergistically increased) anti-cancer immune response compared to either the corresponding anti-PD-1 Fab (or corresponding anti-PD-1 antibody) alone and / or the corresponding IL-2 protein alone, e.g., a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or greater increase in the anti-cancer immune response compared to each component alone. In certain embodiments, the activating anti-PD-1 Fab / IL-2 protein stimulates increased (e.g., synergistically increased) cancer cell killing activity compared to either the corresponding anti-PD-1 Fab (or corresponding anti-PD-1 antibody) alone and / or the corresponding IL-2 protein alone, e.g., a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or greater increase in cancer cell killing activity compared to each component alone.

[0170] In certain embodiments, the activated anti-PD-L1 Fab / IL-2 protein stimulates an increased (e.g., synergistically increased) anti-cancer immune response compared to either the corresponding anti-PD-L1 Fab (or corresponding anti-PD-L1 antibody) alone and / or the corresponding IL-2 protein alone, for example, a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or greater increase in the anti-cancer immune response compared to each component alone. In certain embodiments, the activated anti-PD-L1 Fab / IL-2 protein stimulates increased (e.g., synergistically increased) cancer cell killing activity compared to either the corresponding anti-PD-L1 Fab (or corresponding anti-PD-L1 antibody) alone and / or the corresponding IL-2 protein alone, e.g., a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or greater increase in cancer cell killing activity compared to each component alone.

[0171] In some embodiments, the disease is cancer, i.e., a subject in need thereof has or is suspected of having cancer. Accordingly, certain embodiments include a method of treating cancer, ameliorating the symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject at least one activatable proprotein described herein. In certain embodiments, the cancer is a primary cancer or a metastatic cancer. In specific embodiments, the cancer is selected from one or more of melanoma (optionally metastatic melanoma), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0172] In some embodiments, as noted above, the cancer is a metastatic cancer. In addition to the above cancers, exemplary metastatic cancers include, among others, without limitation, the following: bladder cancer that has metastasized to the bone, liver, and / or lung; breast cancer that has metastasized to the bone, brain, liver, and / or lung; colorectal cancer that has metastasized to the liver, lung, and / or peritoneum; kidney cancer that has metastasized to the adrenal gland, bone, brain, liver, and / or lung; lung cancer that has metastasized to the adrenal gland, bone, brain, liver, and / or other lung sites; melanoma that has metastasized to the bone, brain, liver, lung, and / or skin / muscle; ovarian cancer that has metastasized to the liver, lung, and / or peritoneum; pancreatic cancer that has metastasized to the liver, lung, and / or peritoneum; prostate cancer that has metastasized to the adrenal gland, bone, liver, and / or lung; gastric cancer that has metastasized to the liver, lung, and / or peritoneum; thyroid cancer that has metastasized to the bone, liver, and / or lung; and uterine cancer that has metastasized to the bone, liver, lung, peritoneum, and / or vagina.

[0173] In certain embodiments, as noted herein, cancer expresses or overexpresses PD-L1. PD-L1 expression levels in tissue (e.g., cancer tissue) samples can be determined by any of a variety of methods. For example, PD-L1 protein levels can be determined by immunohistochemistry (IHC), including chromogenic or fluorescent IHC, enzyme-linked immunosorbent assay (ELISA), or Western blot against human AR protein or gene, among other assays. PD-L1 mRNA levels can be measured by, for example, RT-PCR, such as quantitative competitive (QC) RT-PCR, among other techniques known in the art. Thus, certain embodiments include determining, detecting, or measuring PD-L1 levels in tissue samples from subjects in need thereof. Also included is a step of comparing the PD-L1 levels in the tissue sample with those of a control or reference. Certain embodiments include determining the level of PD-L1 in a sample of cancer tissue from a subject (e.g., biopsy tissue), and administering an activatable proprotein homodimer if the cancer tissue from the subject expresses or overexpresses PD-L1.

[0174] The method for treating cancer can be combined with other therapeutic modalities.For example, the combination therapy described herein can be administered to the subject before, during or after other therapeutic interventions, including symptomatic care, radiation therapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof.Symptomatic care includes the administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment and vomiting, and the administration of anticonvulsants to reduce epileptic seizures.Radiation therapy includes whole brain irradiation, fractionated radiation therapy, and radiosurgery, for example, stereotactic radiosurgery, which can be further combined with conventional surgery.

[0175] Accordingly, certain embodiments include combination therapies for treating cancer, including methods of treating cancer, ameliorating symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject at least one activatable proprotein described herein in combination with at least one additional agent, e.g., a chemotherapeutic agent, a hormone therapy agent, and / or a kinase inhibitor. In some embodiments, administering at least one activatable proprotein enhances the sensitivity of the cancer to an additional agent (e.g., a chemotherapeutic agent, a hormone therapy agent, and / or a kinase inhibitor) by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to the additional agent alone.

[0176] Certain combination therapies utilize one or more chemotherapeutic agents, e.g., small molecule chemotherapeutic agents, non-limiting examples of which include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), anti-microtubule agents, among others.

[0177] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mytomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine).

[0178] Examples of antimetabolites include antifolates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0179] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0180] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0181] Those skilled in the art will understand that the various chemotherapeutic agents described herein can be combined with any one or more of the activatable proproteins described herein and used in accordance with any one or more of the methods or compositions described herein.

[0182] Some combination therapy uses at least one hormone therapy agent.General examples of hormone therapy agent include hormone agonist and hormone antagonist.Specific examples of hormone agonist include progestogen (progestin), corticosteroid (for example, prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factor (for example, VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen and somatostatin analogue. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including their analogs. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs; e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0183] Also included are hormone pathway inhibitors, such as antibodies against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alacizumab pegol, bevacizumab, icrucumab, ramucirumab; inhibitors of TGF-beta receptors R1, R2, and R3, such as fresolimumab and metelimumab; inhibitors of c-Met, such as naxitamab; inhibitors of EGF receptors, such as cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, and zalutumumab; inhibitors of FGF receptors, such as aprutumab ixadotin and bemarituzumab; and inhibitors of PDGF receptors, such as olaratumab and tovetumab.

[0184] Those skilled in the art will understand that the various hormone therapy agents described herein can be combined with any one or more of the various activatable proproteins described herein and used according to any one or more of the methods or compositions described herein.

[0185] Certain combination therapies utilize at least one kinase inhibitor, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include, without limitation, adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, and rivaroxaban. itinib), gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0186] One of ordinary skill in the art will understand that the various kinase inhibitors described herein can be combined with any one or more of the various activatable proproteins described herein and used according to any one or more of the methods or compositions described herein.

[0187] In some embodiments, the methods and pharmaceutical compositions described herein increase the subject's median survival time by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks or more.In certain embodiments, the methods and pharmaceutical compositions described herein increase the subject's median survival time by 1 year, 2 years, 3 years or more.In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more.In certain embodiments, the methods and pharmaceutical compositions described herein increase progression-free survival by 1 year, 2 years, 3 years or more.

[0188] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause tumor regression, as indicated by a statistically significant reduction in the amount of viable tumor, for example, a reduction of at least 10%, 20%, 30%, 40%, 50% or more in tumor volume, or by altered (e.g., statistically significantly reduced) scan dimensions. In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause stable disease.

[0189] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to produce a clinically relevant reduction in the symptoms of a particular disease indication known to a skilled clinician.

[0190] For in vivo use, as noted above, for the treatment or testing of human or non-human mammalian disease, the agents described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions, including veterinary therapeutic compositions, prior to administration.

[0191] Thus, certain embodiments relate to pharmaceutical or therapeutic compositions comprising at least one activatable proprotein described herein. In some examples, the pharmaceutical or therapeutic composition comprises one or more of the activatable proproteins described herein in combination with a pharmaceutically or physiologically acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent, such as a chemotherapeutic agent, a hormone therapy agent, and / or a kinase inhibitor described herein.

[0192] Some therapeutic compositions include (and certain methods utilize) only one activatable proprotein. Certain therapeutic compositions include (and certain methods utilize) a mixture of at least two, three, four, or five different activatable proproteins.

[0193] In certain embodiments, pharmaceutical or therapeutic compositions comprising at least one activatable proprotein are substantially pure on a protein basis or weight-by-weight basis, e.g., the compositions have a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis or weight-by-weight basis.

[0194] In some embodiments, the activatable proproteins described herein do not form aggregates, have desirable solubility, and / or have an immunogenicity profile suitable for human use, as known in the art. Thus, in some embodiments, therapeutic compositions comprising an activatable proprotein are substantially aggregate-free. For example, certain compositions contain less than about 10% (on a protein basis) of high molecular weight aggregated protein, or less than about 5% of high molecular weight aggregated protein, or less than about 4% of high molecular weight aggregated protein, or less than about 3% of high molecular weight aggregated protein, or less than about 2% of high molecular weight aggregated protein, or less than about 1% of high molecular weight aggregated protein. Some compositions comprise an activatable proprotein that is at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% monodisperse with respect to its apparent molecular mass.

[0195] In some embodiments, the activatable proprotein is at or near about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14, or 15 mg / ml or at least about 0. Concentrated to 1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14 or 15 mg / ml and formulated for biotherapeutic use.

[0196] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more agents is mixed with any pharmaceutical carrier(s) or excipient(s) known to those skilled in the art to be appropriate for the particular agent and / or mode of administration. Pharmaceutical carriers can be liquid, semi-liquid, or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can contain, for example, sterile diluents (e.g., water), saline solutions (e.g., phosphate-buffered saline; PBS), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents (e.g., benzyl alcohol and methylparabens); antioxidants (e.g., ascorbic acid and sodium bisulfite) and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetates, citrates, and phosphates). For intravenous administration (e.g., via IV infusion), suitable carriers include solutions containing physiological saline or phosphate-buffered saline (PBS), as well as thickeners and stabilizers, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0197] The administration of the agents described herein, whether in pure form or in suitable therapeutic or pharmaceutical compositions, can be carried out via any of the accepted modes of administration of drugs to provide similar benefits.Therapeutic or pharmaceutical compositions can be prepared by combining the drug-containing composition with suitable physiologically acceptable carriers, diluents or excipients, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols.In addition, other pharmaceutically active ingredients (including other small molecules described elsewhere herein) and / or suitable excipients, such as salts, buffers and stabilizers, can be present in the composition, but do not need to be.

[0198] Administration can be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. The preferred mode of administration depends on the nature of the condition being treated or prevented. Certain embodiments include administration by IV infusion.

[0199] Carriers can include, for example, pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Often, a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as polysorbate 20 (TWEEN®), polyethylene glycol (PEG), and poloxamer (PLURONICS®).

[0200] In some embodiments, one or more agents may be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes may further comprise other therapeutic or diagnostic agents.

[0201] The exact dosage and duration of treatment are a function of the disease being treated, and can be determined experimentally using known test protocols or by testing the composition in a model system known in the art and extrapolating from there.Controlled clinical trials can also be carried out.Dosage can also vary depending on the severity of the condition to be alleviated.Pharmaceutical compositions are generally formulated and administered to exert therapeutically useful effects while minimizing undesirable side effects.The composition can be administered at once or divided into several smaller doses that are administered at intervals.For any particular subject, the specific dosage regimen can be adjusted over time according to individual requirements.

[0202] Thus, typical routes of administration of these and related therapeutic or pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, and intrasternal injection or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a subject or patient. The composition administered to a subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the medication described herein in aerosol form may hold multiple dosage units. Actual methods for preparing such dosage forms are known or apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The compositions to be administered typically contain a therapeutically effective amount of an agent described herein for treating the disease or condition of interest.

[0203] The therapeutic or pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier(s) are granular, so that the composition is, for example, in tablet or powder form. The carrier(s) may be liquid, so that the composition is, for example, an oral oil, an injectable liquid, or an aerosol, useful, for example, in inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the forms considered herein as either solid or liquid. Certain embodiments include sterile injectable solutions.

[0204] As a solid composition for oral administration, pharmaceutical compositions can be formulated into powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate, or orange flavoring; and colorings. When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it can contain, in addition to the above-mentioned materials, a liquid carrier, such as polyethylene glycol or oil.

[0205] Therapeutic or pharmaceutical compositions can be in the form of liquid, such as elixir, syrup, solution, emulsion or suspension.Liquid can be for oral administration or for delivery by injection, as two examples.When intended for oral administration, preferred compositions contain one or more of the following: sweetener, preservative, dye / coloring agent and flavor enhancer, in addition to the compound of the present invention.In the composition intended for administration by injection, one or more of surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can be included.

[0206] Liquid therapeutic or pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, as well as agents for adjusting osmotic pressure, such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0207] Liquid therapeutic or pharmaceutical compositions intended for either parenteral or oral administration should contain an amount of agent such that an appropriate dosage is obtained. Typically, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount can be varied to be between 0.1% and about 70% by weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01% and 10% by weight of the agent of interest before dilution.

[0208] The therapeutic or pharmaceutical composition may be intended for external administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.For example, the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.A thickener may be present in the therapeutic or pharmaceutical composition for external administration.When intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

[0209] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example, in the form of suppositories that melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0210] Therapeutic or pharmaceutical compositions may contain various materials that modify the physical form of solid or liquid dosage units. For example, the composition may contain a material that forms a coating shell around the active ingredient. The material that forms the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may contain a component that binds to the drug and thereby aids in the delivery of the compound. Suitable components that can act in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0211] Therapeutic or pharmaceutical compositions can consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to refer to a variety of systems, ranging from systems of colloidal nature to systems consisting of pressurized packaging. Delivery can be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient(s). Aerosol delivery includes the necessary containers, activators, valves, subcontainers, etc., which can be combined to form a kit. One of ordinary skill in the art can determine a preferred aerosol without undue experimentation.

[0212] The compositions described herein can be prepared with carriers that protect the agent against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include, for example, implants and microencapsulated delivery systems, and sustained-release formulations made from biodegradable, biocompatible polymers, such as, but not limited to, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others known to those skilled in the art.

[0213] Therapeutic or pharmaceutical compositions can be prepared by the methodology well known in the pharmaceutical field.For example, therapeutic or pharmaceutical compositions intended to be administered by injection can contain one or more of salts, buffers and / or stabilizers together with sterile distilled water to form a solution.Surfactants can be added to facilitate the formation of a uniform solution or suspension.Surfactants are compounds that interact non-covalently with drugs to facilitate the dissolution or uniform suspension of drugs in aqueous delivery systems.

[0214] Therapeutic or pharmaceutical compositions may be administered in therapeutically effective amounts that vary depending on a variety of factors, including the activity of the particular compound used, the metabolic stability and duration of action of the compound, the age, weight, general health, sex, and diet of the subject, the mode and time of administration, the rate of excretion, the drug combination, the severity of the particular disorder or condition, and the subject being treated. In some examples, the therapeutically effective daily dose is about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g) (for a 70 kg mammal); preferably, the therapeutically effective dose is about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g) (for a 70 kg mammal); more preferably, the therapeutically effective dose is about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g) (for a 70 kg mammal). In some embodiments, the therapeutically effective dose is administered on a weekly, biweekly, or monthly basis. In specific embodiments, the therapeutically effective dose is administered at a dose of, for example, about 1-10 or 1-5 mg / kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg, on a weekly, biweekly, or monthly basis.

[0215] The combination therapy described herein can include the administration of a single pharmaceutical dosage formulation containing an activatable proprotein and an additional therapeutic agent (e.g., a chemotherapeutic agent, a hormone therapy agent, a kinase inhibitor), as well as the administration of a composition containing an activatable proprotein and an additional therapeutic agent in their own separate pharmaceutical dosage formulations. For example, the activatable proprotein and the additional therapeutic agent can be administered to a subject together in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation. Similarly, the activatable proprotein and the additional therapeutic agent can be administered to a subject together in a single parenteral dosage composition, for example, in saline solution or other physiologically acceptable solution, or each agent can be administered in a separate parenteral dosage formulation. As another example, for cell-based therapy, the activatable proprotein can be mixed with the cells prior to administration, administered as a separate composition, or both. When separate dosage formulations are used, the compositions can be administered essentially simultaneously, i.e., concurrently, or at staggered times, i.e., sequentially, and in any order; combination therapy is understood to include all of these regimens.

[0216] Also included are patient care kits that include (a) at least one activatable proprotein described herein; and, optionally, (b) at least one additional therapeutic agent (e.g., a chemotherapeutic agent, a hormone therapy agent, a kinase inhibitor). In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0217] The kits herein may also include one or more additional therapeutic agents or other components appropriate or desired for the indication being treated or for the desired diagnostic application. The kits herein may also include one or more syringes or other components (e.g., stents, implantable depots, etc.) necessary or desired to facilitate the intended mode of delivery.

[0218] In some embodiments, the patient care kit contains separate containers, dividers, or compartments for the composition(s) and informational material(s). For example, the composition(s) can be contained in a bottle, vial, or syringe, and the informational material(s) can be contained in association with the container. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe having the informational material in the form of a label attached thereto. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the activatable proprotein and, optionally, at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the activatable proprotein and, optionally, at least one additional therapeutic agent. The containers of the kit can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0219] The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a metered dose of the agent(s). Also included are methods of providing a kit, for example, by combining the components described herein. Expression and purification system

[0220] Certain embodiments include methods and related compositions for expressing and purifying the activatable proproteins described herein. Such recombinant activatable proproteins can be conveniently prepared using standard protocols, such as those described in Sambrook, et al. (1989, supra), especially sections 16 and 17; Ausubel et al. (1994, supra), especially chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997), especially chapters 1, 5, and 6. As a general example, the activatable proprotein may be prepared by one or more of the following procedures: (a) preparing one or more vectors or constructs comprising one or more polynucleotide sequences encoding the first and second polypeptides described herein and the VL / CL regions of the anti-PD-1 or anti-PD-L1 Fab regions described herein, operably linked to one or more regulatory elements; (b) introducing the one or more vectors or constructs into one or more host cells; (c) culturing the one or more host cells so as to express the first and second polypeptides and the VL / CL regions that combine together to form the activatable proprotein; and (d) isolating the activatable proprotein from the host cells.

[0221] To express the desired polypeptide, the nucleotide sequence encoding the first and / or second polypeptide chain of the activatable proprotein can be inserted into an appropriate expression vector(s), i.e., a vector(s) containing the necessary elements for transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct an expression vector containing a sequence encoding the desired polypeptide and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0222] A variety of expression vector / host systems are known and can be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells, more particularly human cell lines.

[0223] "Control elements" or "regulatory sequences" present in an expression vector are untranslated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—that interact with host cell proteins to effect transcription and translation. Such elements vary in their strength and specificity. Depending on the vector system and host utilized, any number of appropriate transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning into bacterial systems, inducible promoters may be used, such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.). In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. When it is necessary to generate a cell line containing multiple copies of a polypeptide-encoding sequence, SV40- or EBV-based vectors may be advantageously used with an appropriate selectable marker.

[0224] In bacterial systems, several expression vectors can be selected depending on the intended use for the expressed polypeptide. For example, if large quantities are needed, vectors that direct high-level expression of easily purified fusion proteins can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene); pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)), into which a sequence encoding a polypeptide of interest can be ligated in frame with the sequence for the amino-terminal Met and subsequent 7 residues of β-galactosidase to produce a hybrid protein. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be engineered to contain heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0225] Certain embodiments use E. coli-based expression systems (see, e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments may rely in part or in whole on ligation-independent cloning (LIC) to generate suitable expression vectors. In specific embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., the pET vector series). These and related embodiments may utilize the expression host strain BL21(DE3), a lambda DE3 lysogen of BL21 that supports T7-mediated expression and is deficient in lon and ompT proteases for improved target protein stability. Also included are expression host strains harboring plasmids encoding tRNAs rarely used in E. coli, such as the ROSETTA™(DE3) and Rosetta 2(DE3) strains. Cell lysis and sample handling can also be improved using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® protein extraction reagent. For cell culture, autoinducing media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of media (e.g., the OVERNIGHT EXPRESS™ Autoinduction System) gradually initiates protein expression via a metabolic shift without the addition of artificial inducers such as IPTG. Certain embodiments use a hexahistidine tag (e.g., one sold under the trademark HIS·TAG® fusion) followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in certain aspects, clinical-grade proteins can be isolated from E. coli inclusion bodies with or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).As a further example, overexpression of proteins in Escherichia coli at low temperatures improves their solubility and stability, so certain embodiments may use a cold-shock-induced high-yield production system in E. coli (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0226] High-density bacterial fermentation systems are also included. For example, high-cell-density cultivation of Ralstonia eutropha allows protein production at cell densities of over 150 g / L and expression of recombinant proteins at titers of over 10 g / L.

[0227] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544 (1987). Pichia pandoris expression systems are also included (see, for example, Li et al., Nature Biotechnology. 24, 210-215, 2006; and Hamilton et al., Science, 301:1244, 2003). Certain embodiments include yeast systems engineered to selectively glycosylate proteins, including, inter alia, yeast with humanized N-glycosylation pathways (see, e.g., Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature-Biotechnology. 22:1409-1414, 2004; U.S. Patent Nos. 7,629,163; 7,326,681; and 7,029,872). By way of example only, recombinant yeast cultures can be grown in Fernbach flasks or 15 L, 50 L, 100 L, and 200 L fermentors, among others.

[0228] When using a plant expression vector, the expression of the sequence encoding the polypeptide can be driven by any of several promoters. For example, viral promoters, such as the 35S and 19S promoters of CaMV, can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters, such as the small subunit of RUBISCO or heat shock promoters, can be used (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several publicly available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0229] Insect systems can also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia cells. A polypeptide-encoding sequence can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence renders the polyhedrin gene inactive and produces a recombinant virus lacking coat protein. The recombinant virus can then be used to infect, for example, S. frugiperda cells or Trichoplusia cells in which the polypeptide of interest can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)). Baculovirus expression systems are also included, including those utilizing SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5:Unit 5.4, 2001). Insect systems can provide post-translational modifications similar to mammalian systems.

[0230] In mammalian host cells, several virus-based expression systems are commonly available. For example, when adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest can be ligated into the adenovirus transcription / translation complex consisting of a late promoter and a tripartite leader sequence. Insertion into the non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0231] Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines, such as NSO and Sp2 / 0. For a review of certain mammalian host cell systems suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems.Mammalian expression systems can utilize, for example, T-flasks, roller bottles, or cell factories, or attached cell lines in suspension culture, for example, in 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L, and 200 L stirred tank bioreactors, or 20 / 50 L and 100 / 200 L WAVE bioreactors, among others known in the art.

[0232] Cell-free expression of proteins is also included. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides; these reagents may be produced by extraction from cells or from cell-based expression systems.

[0233] Specific initiation signals can also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. When a polypeptide-encoding sequence, its initiation codon, and upstream sequences are inserted into an appropriate expression vector, additional transcriptional or translational control signals may not be required. However, if only a coding sequence or a portion thereof is inserted, exogenous translational control signals, including the ATG initiation codon, should be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by including enhancers appropriate for the particular cell system used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0234] Furthermore, host cell lines can be selected for their ability to modulate the expression of inserted sequences or process expressed proteins in the desired manner. Such modifications of polypeptides include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "pre-pro" form of the protein can also be used to facilitate correct insertion, folding, and / or function. In addition to bacterial cells, different host cells, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, which have or lack specific cellular machinery and characteristic mechanisms for such post-translational activities, can be selected to ensure the correct modification and processing of foreign proteins.

[0235] Stable expression is generally preferred for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing a polynucleotide of interest can be transformed using expression vectors that may contain a viral origin of replication and / or endogenous expression elements and a selectable marker gene on the same or separate vectors. After introduction of the vector, cells can be grown in an enriched medium for approximately 1-2 days before being switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection; its presence allows growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type. Transient production, for example, by transient transfection or infection, can also be used. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0236] A number of selection systems can be used to recover transformed or transduced cell lines, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be used in tk- or aprt- cells, respectively. Additionally, the following antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection: for example, dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)); npt, which confers resistance to the aminoglycosides, neomycin, and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat, which confers resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85:8047-51 (1988)). The use of visible markers has become popular, with markers such as green fluorescent protein (GFP) and other fluorescent proteins (e.g., RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin being widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression attributed to a particular vector system (see, for example, Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0237] High-throughput protein production system or micro-production system is also included.Certain embodiments can utilize hexa-histidine fusion tag for protein expression and purification on metal chelate modified slide surface or MagneHis Ni particle (see, for example, Kwon et al., BMC Biotechnol.9:72,2009; and Lin et al., Methods Mol Biol.498:129-41,2009).High-throughput cell-free protein expression system is also included (see, for example, Sitaraman et al., Methods Mol Biol.498:229-44,2009).

[0238] A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products using product-specific binding agents or antibodies, such as polyclonal or monoclonal antibodies, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Western immunoblot, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0239] A wide variety of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting polynucleotide-related sequences include oligolabeling, nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence or any portion thereof can be cloned into a vector for the production of mRNA probes. Such vectors are known in the art and commercially available, and can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase, such as T7, T3, or SP6, and labeled nucleotides. These procedures can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0240] Host cells transformed with one or more polynucleotide sequences of interest can be cultured under suitable conditions for protein expression and its recovery from cell culture.Certain specific embodiments utilize serum-free cell expression systems.Examples include HEK293 cells and CHO cells that can grow on serum-free medium (see, for example, Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent No. 6,210,922).

[0241] The activatable proprotein produced by a recombinant cell can be secreted or contained intracellularly, depending on the sequence and / or the vector used. As will be understood by those skilled in the art, expression vectors containing polynucleotides can be designed to contain signal sequences that direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to connect a sequence encoding a polypeptide of interest to a nucleotide sequence encoding a polypeptide domain that facilitates purification and / or detection of soluble proteins. Examples of such domains include cleavable and non-cleavable affinity purification and epitope tags, such as avidin, FLAG tags, poly-histidine tags (e.g., 6xHis), cMyc tags, V5 tags, glutathione S-transferase (GST) tags, and the like.

[0242] Proteins produced by recombinant cells can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reverse phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Analytical methods that can be utilized during any step of the production or purification process to typically measure the purity of a protein composition are also included, such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblot, Bradford, and ELISA.

[0243] Also included are methods for concentrating activatable proproteins and compositions comprising concentrated soluble activatable proproteins. In some embodiments, such concentrated solutions of at least one activatable proprotein comprise the protein at a concentration of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or more.

[0244] In some embodiments, such compositions may be substantially monodisperse, meaning that the activatable proprotein is present predominantly (i.e., at least about 90% or more) in one apparent molecular weight form as assessed, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0245] In some aspects, such compositions have a purity (on a protein basis) of at least about 90%, or in some aspects at least about 95%, or in some embodiments at least 98%. Purity can be determined via any conventional analytical method known in the art.

[0246] In some aspects, such compositions have a high molecular weight aggregate content of less than about 10% relative to the total amount of protein present, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 5%, or in some aspects, such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments, less than about 1%. High molecular weight aggregate content can be determined via various analytical techniques, including, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0247] The example of the concentration approach contemplated herein includes freeze-drying, which is typically used when the solution contains almost no soluble components other than the protein of interest.Freeze-drying is often carried out after HPLC, and can remove most or all volatile components from the mixture.Also included is ultrafiltration technique, which typically uses one or more selectively permeable membranes to concentrate protein solution.This membrane allows water and small molecules to pass through, but retains protein; the solution can be pushed against the membrane by mechanical pump, gas pressure or centrifugation, among other techniques.

[0248] In certain embodiments, the activatable proprotein in the composition has a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the activatable proprotein composition has a purity of at least about 95%, or at least about 97%, 98%, or 99%. In some embodiments, for example, when used as a reference or research reagent, the activatable proprotein may be of lower purity and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity may be measured, for example, by the purity of the protein standard, either in total or in relation to selected components, such as other proteins.

[0249] Purified activatable proproteins can also be characterized according to their biological characteristics. Binding affinity and binding kinetics can be measured according to various techniques known in the art, such as Biacore® and related technologies, which utilize surface plasmon resonance (SPR), an optical phenomenon that allows real-time detection of unlabeled interactants. SPR-based biosensors can be used in determining active concentrations, screening, and characterizing both affinity and kinetics. The presence or level of one or more biological activities can be measured according to cell-based assays, including those that utilize at least one IL-2 receptor, optionally functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of biological activity as described herein.

[0250] In certain embodiments, as noted above, the activatable proprotein composition is substantially endotoxin-free, including, for example, about 95% endotoxin-free, preferably about 99% endotoxin-free, and more preferably about 99.99% endotoxin-free. The presence of endotoxin can be detected according to routine techniques in the art, as described herein. In specific embodiments, the activatable proprotein composition is produced from eukaryotic cells, e.g., mammalian or human cells, in a substantially serum-free medium. In certain embodiments, as noted herein, the activatable proprotein composition has an endotoxin content of less than about 10 EU / mg activatable proprotein, or less than about 5 EU / mg activatable proprotein, or less than about 3 EU / mg activatable proprotein, or less than about 1 EU / mg activatable proprotein.

[0251] In certain embodiments, the activatable proprotein composition comprises less than about 10% wt / wt of high molecular weight aggregates, or less than about 5% wt / wt of high molecular weight aggregates, or less than about 2% wt / wt of high molecular weight aggregates, or less than about 1% wt / wt or less than about 1% wt / wt of high molecular weight aggregates.

[0252] Also included are protein-based analytical assays and methods that can be used to evaluate, among other characteristics, protein purity, size, solubility, and degree of aggregation.Protein purity can be evaluated in several ways.For example, purity can be evaluated based on primary structure, higher-order structure, size, charge, hydrophobicity, and glycosylation.Examples of methods for evaluating primary structure include N-terminal and C-terminal sequencing and peptide mapping (see, for example, Allen et al., Biologicals. 24:255-275, 1996). Exemplary methods for assessing conformation include circular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, e.g., Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuterium exchange kinetics, differential scanning calorimetry, NMR spectroscopy, and immunoreactivity with conformationally sensitive antibodies. Conformation can also be assessed as a function of various parameters, such as pH, temperature, or added salt. Exemplary methods for assessing protein characteristics, such as size, include analytical ultracentrifugation and size-exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion-exchange chromatography and isoelectric focusing. Hydrophobicity can be assessed, for example, by reverse-phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetics (eg, clearance), conformation or stability, receptor binding, and protein function and can be assessed, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0253] As noted above, certain embodiments include the use of SEC-HPLC to evaluate protein characteristics, such as purity, size (e.g., size uniformity), or degree of aggregation, and / or to purify proteins, among other uses. SEC, which also includes gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in solution are separated in a porous material based on their size, or more specifically, their hydrodynamic volume, diffusion coefficient, and / or surface properties. This process is commonly used to separate biological molecules and determine the molecular weight and molecular weight distribution of polymers. Typically, a biological or protein sample (e.g., a protein extract produced according to the protein expression methods provided herein and known in the art) is loaded into a selected size-exclusion column with a defined stationary phase (porous material), preferably one that does not interact with the proteins in the sample. In certain aspects, the stationary phase is composed of inert particles packed into a dense three-dimensional matrix within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof. Stationary phase particles typically have small pores and / or channels that allow only molecules below a certain size to enter. Larger particles are therefore excluded from these pores and channels, and their limited interaction with the stationary phase causes them to elute as a "totally excluded" peak at the beginning of the experiment. Smaller molecules that can fit into the pores are removed from the flowing mobile phase, and the time they spend immobilized in the stationary phase pores depends in part on how deeply they penetrate into the pores. Their removal from the mobile phase flow increases the time it takes them to elute from the column, resulting in separation between particles based on differences in their size. A given size exclusion column has a range of molecular weights that it can separate.Overall, molecules larger than the upper limit are not captured by the stationary phase, molecules smaller than the lower limit enter the solid phase completely and elute as a single band, and molecules within the range elute at different rates defined by their properties, e.g., hydrodynamic volume. For examples of these methods in practice with pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

[0254] Protein purity for clinical applications has also been discussed, for example, by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). More recent techniques for analyzing protein purity include, but are not limited to, the LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, which provides high-throughput analysis of protein titer, sizing, and purity analysis. In certain non-limiting embodiments, clinical-grade activatable proproteins can be obtained by, among other methods, utilizing a combination of chromatographic materials in at least two orthogonal steps (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, Eds., Smales and James, Humana Press Inc., 2005). Typically, protein drugs (e.g., activatable proproteins) are substantially endotoxin-free when measured according to techniques known in the art and described herein.

[0255] Protein solubility assays are also included. Such assays can be used, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the selection of buffer(s), and to optimize the selection of activatable proproteins and their variants. Solubility or aggregation can be assessed according to various parameters, including temperature, pH, salt, and the presence or absence of other additives. Examples of solubility screening assays include, without limitation, microplate-based methods that measure protein solubility using turbidity or other measures as an endpoint, high-throughput assays for analysis of purified recombinant protein solubility (see, e.g., Stenvall et al., Biochim Biophys Acta. 1752:6-10, 2005), assays that use structural complementation of genetic marker proteins to monitor and measure protein folding and solubility in vivo (see, e.g., Wigley et al., Nature Biotechnology. 19:131-136, 2001), and electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2006), among others. Activatable proproteins with increased solubility (or reduced aggregation) can be identified or selected according to techniques routine in the art, including simple in vivo assays for protein solubility (see, e.g., Maxwell et al., Protein Sci. 8:1908-11, 1999).

[0256] Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term that encompasses several types of interactions or characteristics, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and native / denatured interactions and characteristics. For protein therapeutics, the presence of aggregates is typically considered undesirable due to concerns that aggregates may cause immunogenic reactions (e.g., small aggregates) or adverse events upon administration (e.g., particulate matter). Dynamic light scattering refers to a technique that can be used to determine the size distribution profile of small particles in suspension or polymers, such as proteins, in solution. This technique, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), uses scattered light to measure the rate of diffusion of protein particles. Fluctuations in the scattering intensity can be observed due to the Brownian motion of molecules and particles in solution. This motion data can be conventionally processed to derive a size distribution for the sample, where the size is given by the Stokes radius or hydrodynamic radius of the protein particles. Hydrodynamic size depends on both mass and shape (conformation). Dynamic light scattering can detect the presence of very small amounts of aggregated protein (<0.01% by weight), even in samples containing a large range of masses. It can also be used to compare the stability of different formulations, including applications that rely on real-time monitoring of changes at elevated temperatures. Thus, certain embodiments include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing the activatable proproteins of the present disclosure.

[0257] While the foregoing embodiments have been described in some detail by way of example and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of the present disclosure that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following examples are offered by way of illustration only, and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Example]

[0258] Example 1A Preparation of PD-L1-proIL-2 fusion protein Plasmids encoding PD-L1-proIL-2v (IL-2 variant), PD-L1-proIL-2wt, or anti-human PD-L1 with different protease-cleavable linkers were constructed by standard gene synthesis and then subcloned into the pTT5 expression vector. Schematics of exemplary IgG-proIL-2 fusion protein formats are shown in Figures 1-2.

[0259] Exemplary proteins in the PD-L1-proIL-2v ("v" = variant) format with different protease cleavable linkers include P40391942 (SEQ ID NOs: 138 and 139), P40531942 (SEQ ID NOs: 140 and 141), and P40621942 (SEQ ID NOs: 142 and 143). Exemplary proteins in the PD-L1-proIL-2wt format include P47131942 (SEQ ID NOs: 154 and 155). Exemplary anti-human PD-L1 proteins include P40751942 (SEQ ID NOs: 144 and 145).

[0260] PD-L1-proIL-2 fusion protein or anti-human PD-L1 was produced by transient transfection in Expi293 cells and purified by one-step MabSelect SuRe chromatography (GE Healthcare). The purified protein was characterized by SDS-PAGE and high-performance liquid chromatography (HPLC) to assess purity and homogeneity. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and an Agilent 1260 according to the manufacturer's instructions. The purified protein demonstrated high purity on SDS-PAGE gels and good homogeneity based on the HPLC results.

[0261] Example 1B Preparation of PD-1-proIL-2v fusion protein Plasmids encoding PD-1-proIL-2v, PD-1-null-proIL-2v, or anti-human PD-1 with different protease-cleavable linkers were constructed by standard gene synthesis and then subcloned into the pTT5 expression vector. Schematics of exemplary IgG-proIL-2 fusion protein formats are shown in Figures 1 and 2.

[0262] Example proteins of the PD-1-proIL-2v format with different protease-cleavable linkers include P41222037 (SEQ ID NOs: 146 and 147), P43492037 (SEQ ID NOs: 152 and 153). Example proteins of the unmasked PD-1-proIL-2v format include P41252037 (SEQ ID NOs: 158 and 159). Example proteins of the PD-1-null-proIL-2v format include P54475445 (SEQ ID NOs: 156 and 157). Example proteins of anti-human PD-1 include P42412037 (SEQ ID NOs: 150 and 151).

[0263] PD-1-proIL-2v or PD-1-null-proIL-2v fusion proteins or anti-human PD-1 were produced by transient transfection in Expi293 cells and purified by one-step MabSelect SuRe chromatography (GE Healthcare). The purified proteins were characterized by SDS-PAGE and high-performance liquid chromatography (HPLC) to assess purity and homogeneity. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and an Agilent 1260 according to the manufacturer's instructions. The purified proteins demonstrated high purity on SDS-PAGE gels and good homogeneity based on the HPLC results.

[0264] Example 1C Preparation of B7H3-proIL-2 fusion protein A plasmid encoding B7H3-proIL-2v was constructed by standard gene synthesis and then subcloned into the pTT5 expression vector. A schematic diagram of an exemplary IgG-proIL-2 fusion protein format is shown in Figures 1-2.

[0265] Exemplary proteins of the B7H3-proIL-2v format include P41713699 (SEQ ID NOs: 148 and 149).

[0266] B7H3-proIL-2v fusion protein was produced by transient transfection in Expi293 cells and purified by one-step MabSelect SuRe chromatography (GE Healthcare). The purified protein was characterized by SDS-PAGE and high-performance liquid chromatography (HPLC) to assess purity and homogeneity. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and an Agilent 1260 according to the manufacturer's instructions. The purified protein showed high purity on SDS-PAGE gels and good homogeneity based on the HPLC results.

[0267] Example 2A Binding of PD-1-proIL-2 to both human PD-1 and cynomolgus monkey PD-1 The binding activity of P41222037 and P42412037 was determined by ELISA. Microtiter plates were coated with 100 μl of 2 μg / ml streptavidin overnight at 4°C. The following day, the plates were washed with PBS and blocked with 2% BSA (2% bovine serum albumin in PBS). 100 μl of 2 μg / ml biotinylated huPD-1 or CynoPD-1 was added to the corresponding wells and incubated at room temperature for 1 hour to capture the biotinylated proteins. The plates were washed sequentially with PBST (0.01% Tween® in PBS) and PBS.

[0268] The indicated samples (PD-1 IgG or PD-1-proIL-2v) used in the ELISA assay were prepared in 2% BSA to an initial concentration of 10 μg / ml and then serially diluted 1 / 3. 100 μl of diluted protein was added to the corresponding wells and incubated at room temperature for 1 hour. The plate was washed sequentially with PBST and PBS.

[0269] Bound antibodies were detected using a peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch). The plates were washed sequentially with PBST and PBS. 90 μl of TMB substrate was added to each well and incubated in the dark at room temperature for 5 minutes. The reaction was terminated with 45 μl of 2 M sulfuric acid, and the absorbance was read at 450 nm. Data were analyzed using Prism.

[0270] As shown in Figures 4A-4B, PD-1-proIL-2v and the corresponding PD-1 IgG bind similarly to human PD-1.

[0271] Example 2B Binding of PD-L1-proIL-2 to human PD-L1 The binding activity of P40391942 and P40751942 was determined by ELISA. Microtiter plates were coated with 100 μl of 2 μg / ml streptavidin overnight at 4°C. The following day, the plates were washed with PBS and blocked with 2% BSA (2% bovine serum albumin in PBS). 100 μl of 2 μg / ml biotinylated huPD-L1 was added to the corresponding wells and incubated at room temperature for 1 hour to capture the biotinylated proteins. The plates were washed sequentially with PBST (0.01% Tween® in PBS) and PBS.

[0272] The indicated samples (PD-L1 IgG or PD-L1-proIL-2v) used in the ELISA assay were prepared in 2% BSA to an initial concentration of 10 μg / ml, then serially diluted 1 / 3. 100 μl of diluted protein was added to the corresponding wells and incubated at room temperature for 1 hour. The plate was washed sequentially with PBST and PBS.

[0273] Bound antibodies were detected using a peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch). The plates were washed sequentially with PBST and PBS. 90 μl of TMB substrate was added to each well and incubated in the dark at room temperature for 5 minutes. The reaction was terminated with 45 μl of 2 M sulfuric acid, and the absorbance was read at 450 nm. Data were analyzed using Prism.

[0274] As shown in Figure 4C, PD-L1-proIL-2v and the corresponding PD-L1 IgG bind similarly to human PD-L1.

[0275] Example 2C PD-1-proIL-2v restores T cell function suppressed by PD-1 Mature DC cells were recovered, treated with 50 μg / ml mitomycin C in the dark for 30 minutes, and washed twice with PBS. Recovered PBMCs (1e5 cells / well) and allogeneic DCs (5000 cells / well) were co-cultured with increasing doses of isotype control antibody, parental PD-1 antibody, or PD-1-proIL-2 fusion protein at an initial concentration of 100 μg / ml. After 5 days of incubation at 37°C, IFNγ secretion in the culture supernatant was analyzed using a human IFNγ ELISA set (Biolegend catalog 430104). Pre-coated capture antibody plates were prepared as described in the manufacturer's instructions, and 100 μl of diluted standard or sample was added to the corresponding wells and incubated at room temperature for 2 hours. The plates were washed four times with wash buffer, and 100 μl of diluted detection antibody solution was added to each well and incubated at room temperature for 30 minutes with shaking. The plate was then washed five times with wash buffer, and 100 μl of freshly mixed TMB substrate solution was added to each well and incubated in the dark at room temperature for 20 minutes. The reaction was terminated with 100 μl of stop solution, and the absorbance was read at 450 nm. Data was analyzed by Prism.

[0276] As shown in Figure 5, mixed lymphocyte reaction assay data demonstrate that PD-1-proIL-2v restores T cell function suppressed by PD-1.

[0277] Example 2D Proliferation of M07-e by PD-1-proIL-2v and PD-L1-proIL-2v Human M-07e cells (expressing human IL-2Rβ / γ) were cultured in RPMI 1640 supplemented with 20% fetal bovine serum (FBS) and 10% 5637 cell culture supernatant. To measure cytokine-dependent cell proliferation, M-07e cells were harvested in their logarithmic growth phase and washed twice with PBS. 90 μl of cell suspension (2 × 10 4Cells / well) were seeded in 96-well plates and incubated in assay medium for cytokine starvation (RPMI 1640 supplemented with 10% FBS) for 4 hours at 37° C. and 5% CO 2 .

[0278] The IL-2, P40391942, or P41222037 protein samples used in the assay were prepared in assay medium to an initial concentration (300 nM for IL-2, 24300 nM for PD-1 / PD-L1-proIL-2v, and 2700 nM for MMP2 protease-activated fusion protein) and then serially diluted 1 / 3. 10 μl of diluted protein was added to the corresponding wells and incubated at 37°C and 5% CO2 for 72 hours. A colorimetric assay using Cell Counting Kit-8 (CCK-8, Dojindo, CK04) was performed to measure the amount of viable cells.

[0279] Figure 6A shows that PD-1-proIL-2v fails to induce M07-e cell proliferation, even at the highest concentration. MMP-2-cleaved PD-1-proIL-2v restored partial IL-2 activity, exhibiting approximately 30-fold lower activity compared to wild-type IL-2. Figure 6B shows that PD-L1-proIL-2v had little activity at concentrations as high as 810 nM. MMP-2 protease-activated PD-L1-proIL-2v induced M07-e cell proliferation as efficiently as protease-activated PD-1-proIL-2v.

[0280] Example 3A PBMC cell activation by PD-1-proIL-2v (pSTAT5 assay) Frozen human PBMCs (SAILYBIO, donor 1) were allowed to recover in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin for approximately 2 hours at 37°C in a 5% CO atmosphere. PBMCs were cultured at a concentration of 2.6 x 10 6Adjust to cells / ml and inoculate 4 x 10 cells per well in a 96-well plate. 5 Cells were plated. Recovered PBMCs were incubated with rhIL-2, P41222037, or P54475445 for 15 minutes at 37°C. After incubation, cells were immediately fixed with Cytofix buffer (BD Bioscience), phosphorylation status was measured, and cells were stored on ice for 15 minutes and washed once with BD Pharmingen™ staining buffer (FBS).

[0281] For surface staining, cells were incubated with CD3 Alexa Flour 700 (BD 557943), CD4 PerCP-Cytm5.5 (BD 560650), CD8 APC-Cytm7 (BD 557760), CD25 BV421 (BD 562442), and CD56 BV510 (BD 744218) for 30 minutes at 4°C. Cells were washed once with 1x PBS, centrifuged at 500g for 5 minutes, and the supernatant was removed by aspiration. Cells were permeabilized with pre-chilled Phosflow Perm Buffer III (BD Bioscience) for 30 minutes at 4°C. Before starting intracellular staining, cells were washed once with 1x PBS and centrifuged at 500g for 5 minutes to collect the pellet. Cells were then stained with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa Fluor® 647 (BD 562076) for 40 minutes at room temperature. Cells were washed twice with BD Pharmingen™ staining buffer (FBS) and centrifuged at 500 g for 5 minutes to pellet the cells, and the supernatant was removed. Cell pellets were resuspended in 200 μl (per well) of BD Pharmingen staining buffer (FBS) and analyzed by flow cytometry. STAT5 phosphorylation status in PBMC subsets upon prodrug treatment was acquired and processed by CytoFLEX (Beckman).

[0282] Figures 7A-7D show STAT phosphorylation in CD4 T cells (7A), CD8 T cells (7B), regulatory T cells (7C), and NK cells (7D) upon treatment of resting PBMCs from donor 1 with rhIL-2 and intact and protease-activated PD-1-proIL-2v. The masked or inactive procytokine form of P41222037 failed to induce STAT5 phosphorylation in all cell subsets tested. The protease-activated form of P41222037 (cleaved by matrix metalloproteinase-2) was equally effective in activating STAT5 phosphorylation in CD8 and CD4 T cells but was less potent in NK cells. In Treg cells, activated P41222037 was 30-fold less potent than huIL-2. Thus, compared with wild-type huIL-2, P41222037 no longer preferentially activated Treg cells. Notably, prior blockade of the PD-1 receptor with a parent anti-PD-1 antibody reduced the potency of P41222037 (activated form) against PD-1-expressing T cells by more than 20-fold. Similarly, P54475445 (PD-1 null-IL-2v fusion protein) showed 20-fold lower potency against CD4+ T cells, CD8+ T cells, and Treg cells. These data demonstrate that preferential cis-targeting of PD-1-proIL-2v results in enhanced potency against PD-1-positive T cells.

[0283] Example 3B PBMC cell activation by PD-L1-proIL-2v (pSTAT5 assay) Frozen human PBMCs (SAILYBIO, donor 1) were allowed to recover in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin for approximately 2 hours at 37°C in a 5% CO atmosphere. PBMCs were cultured at a concentration of 2.6 x 10 6 Adjust to cells / ml and inoculate 4 x 10 cells per well in a 96-well plate. 5Recovered PBMCs were plated with rhIL-2 and P40391942 (both intact and activated forms) for 15 minutes at 37°C. After incubation, cells were immediately fixed with Cytofix buffer (BD Bioscience), phosphorylation status was monitored, and cells were stored on ice for 15 minutes and washed once with BD Pharmingen™ staining buffer (FBS).

[0284] For surface staining, cells were incubated with CD3 Alexa Flour 700 (BD 557943), CD4 PerCP-Cytm5.5 (BD 560650), CD8 APC-Cytm7 (BD 557760), CD25 BV421 (BD 562442), and CD56 BV510 (BD 744218) for 30 minutes at 4°C. Cells were washed once with 1x PBS, centrifuged at 500g for 5 minutes, and the supernatant was removed by aspiration. Cells were permeabilized with pre-chilled Phosflow Perm Buffer III (BD Bioscience) for 30 minutes at 4°C. Before starting intracellular staining, cells were washed once with 1x PBS and centrifuged at 500g for 5 minutes to collect the pellet. Cells were then stained with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa Fluor® 647 (BD 562076) for 40 minutes at room temperature. Cells were washed twice with BD Pharmingen™ staining buffer (FBS) and centrifuged at 500 g for 5 minutes to pellet the cells, and the supernatant was removed. Cell pellets were resuspended in 200 μl (per well) of BD Pharmingen staining buffer (FBS) and analyzed by flow cytometry. STAT5 phosphorylation status in PBMC subsets upon procytokine treatment was acquired and processed by CytoFLEX (Beckman).

[0285] Figures 8A-8D show STAT phosphorylation in CD4 T cells (8A), CD8 T cells (8B), regulatory T cells (8C), and NK cells (8D) upon treatment of resting PBMCs from donor 1 with rhIL-2 and intact and activated PD-L1-proIL-2v. The masked form of P40391942 failed to induce STAT5 phosphorylation in all cell subsets tested. The activated form of P40391942 (cleaved by matrix metalloproteinase-2) was equally effective in activating STAT5 phosphorylation in CD8 and CD4 T cells, but was less potent in NK cells. In Treg cells, activated P40391942 was less potent than huIL-2. Notably, prior blockade of the PD-L1 receptor with a parental anti-PD-L1 antibody reduced the potency of P40391942 (activated form) against PD-L1 expressed on CD4+ T cells, CD8+ T cells, and Treg cells by more than 100-fold. These data demonstrate that PD-L1 binding enhances IL-2v potency.

[0286] Example 3C Activated PD-1-proIL-2v stimulates GM-CSF secretion by human CD4 T cells Frozen PBMCs were recovered and treated with 10 μg / mL mitomycin C for 2 hours in the dark. After three washes, recovered PBMCs were plated at 4e5 cells / well in 96-well plates with increasing concentrations of parental PD-1 antibodies or PD-1-proIL-2 fusion proteins (both intact and activated forms). Frozen CD4 T cells from the same donor were then suspended in RPMI1640 supplemented with 1 μg / mL soluble αCD3; 4e+5 cells / well of CD4 T cells were seeded into the PBMC 96-well plates.

[0287] After 5 days of incubation at 37°C, GM-CSF secretion in the culture supernatant was analyzed using a human GM-CSF ELISA set (Biolegend catalog 432004). Pre-coated capture antibody plates were prepared as described in the manufacturer's instructions, and 100 μl of diluted standard or sample was added to the corresponding wells and incubated at room temperature for 2 hours. The plates were washed four times with wash buffer, and 100 μl of diluted detection antibody solution was added to each well and incubated at room temperature for 30 minutes with shaking. The plates were then washed five times with wash buffer, and 100 μl of freshly mixed TMB substrate solution was added to each well and incubated at room temperature in the dark for 20 minutes. The reaction was terminated with 100 μl of stop solution, and absorbance was read at 450 nm. Data were analyzed using Prism.

[0288] As shown in Figure 9A, protease-activated PD-1-proIL-2v, but not the intact procytokine, stimulates GM-CSF production by CD4 T cells. In contrast, PD-1 blockade alone did not induce any significant levels of GM-CSF secretion. Example 3D Activated PD-1-proIL-2v stimulates IFNγ production in human CD4 T cells

[0289] Frozen PBMCs were recovered and treated with 10 μg / mL mitomycin C for 2 hours in the dark. After three washes, recovered PBMCs were plated at 4e5 cells / well in 96-well plates with increasing concentrations of parental PD-1 antibodies or PD-1-proIL-2 fusion proteins (both intact and activated forms). Frozen CD4 T cells from the same donor were then suspended in RPMI1640 supplemented with 1 μg / mL soluble αCD3; 4e5 cells / well of CD4 T cells were seeded into the PBMC 96-well plates.

[0290] After 5 days of incubation at 37°C, cells were collected for intracellular FACS staining. Cytokine accumulation in the Golgi complex was induced by restimulating the cells with ionomycin (500 ng / ml) and PMA (50 ng / ml) together with protein transport inhibitors (GolgiPlug and GolgiStop, BD) for 5 hours. After incubation, cells were immediately fixed with Cytofix buffer (BD Bioscience) and stored on ice for 15 minutes to determine phosphorylation status. Then, cells were washed once with BD Pharmingen™ staining buffer (FBS). For surface staining, cells were incubated with CD3 Alexa Flour 700 (BD 557943) and CD4 PerCP-Cytm5.5 (BD 560650) for 30 minutes at 4°C. Cells were washed once with 1x PBS, centrifuged at 500g for 5 minutes, and the supernatant was removed by aspiration. Cells were permeabilized with pre-chilled Phosflow Perm Buffer III (BD Bioscience) for 30 minutes at 4°C. Before starting intracellular staining, cells were washed once with 1x PBS and centrifuged at 500g for 5 minutes to collect the pellet. Cells were then stained with BV605 anti-human IFN-γ (BD 562974) for 40 minutes at RT. Cells were washed twice with BD Pharmingen™ staining buffer (FBS) and centrifuged at 500g for 5 minutes to pellet the cells, and the supernatant was removed. The cell pellet was resuspended in 200ul (per well) of BD Pharmingen staining buffer (FBS) and analyzed using a CytoFLEX (Beckman).

[0291] As shown in Figure 9B, activated PD-1-proIL-2v, but not the intact procytokine or the PD-1 antibody alone, stimulates IFNγ production by CD4 T cells.

[0292] Example 4 In vivo efficacy of PD-L1-proIL-2v and PD-1-proIL-2v with different protease-cleavable linkers in a tumor-PBMC xenograft model PD-L1-proIL-2v and PD-1-proIL-2v with different protease-cleavable linkers were tested for their anti-tumor efficacy in tumor-PBMC xenograft models, alone and compared with the corresponding PD-L1 or PD-1 antibodies.

[0293] A375 cells were maintained in vitro in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air. Human PBMCs were cocultured with mitomycin C-treated A375 tumor cells for 6 days and maintained in vitro as suspensions cultured in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air.

[0294] HT29 cells were maintained in vitro in McCoy's 5A supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air. Human PBMCs were cocultured with mitomycin C-treated HT29 tumor cells for 6 days and maintained in vitro as suspensions cultured in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air.

[0295] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8–10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark daily cycle. Mice were maintained in individual ventilated cages at a constant temperature (20–26°C) and humidity (40–70%) with ≤6 animals per cage. Animals had free access to radiation-sterilized dry granular food and sterile drinking water throughout the study period. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva prior to implementation. After arrival, animals were maintained for at least 3 days to acclimate to their new environment. During daily monitoring, animals were checked for any effect of tumor growth on normal behavior, such as mobility, food and water intake (visual observation only), weight gain / loss (body weight was measured twice a week), eye / hair matting, and any other abnormal effects.

[0296] Mice were implanted in the right flank with 4 × 10 cells in 0.2 ml of HBSS (1:1) containing 0.1 ml of Matrigel for tumor development. 6 A375 cells and 4 x 10 5 hPBMCs (co-cultured with A375) or 5 × 10 6 HT29 cells and 1 x 10 6 Mice were subcutaneously inoculated with 1000 mg of hPBMCs (co-cultured with HT29). One week after tumor cell inoculation, mice were injected intravenously with PD-L1-proIL-2v, PD-1-proIL-2v, or PD-L1 and PD-1 antibodies. Mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2where a and b are the longest and shortest diameters of the tumor, respectively. The tumor volume was then used to calculate TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations: TGI%=(1-(T n -T0) / (V n -V0))×100% T / C%=(T n / T0) / (V n / V0)×100%

[0297] In the formula, T n and V n indicates the tumor volume of the treatment group and the vehicle control group, respectively, on the nth day after the start of treatment. TO and V0 indicate the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0298] Because the protease-cleavable linker in P40391942 is the easiest to cleave, and the linker in P40531942 is the most difficult to cleave, Figures 10A-10B and Table 1 show that in an A375-PBMC xenograft model, P40391942 demonstrated the best anti-tumor activity, followed by P40621942 > P40531942 > P40751942 (anti-PD-L1 antibody). 5 / 5 and 2 / 6 mice treated with P40391942 and P40621942, respectively, demonstrated complete responses. [Table 1]

[0299] Figures 11A-11B and Table 2 show that in the HT-29-PBMC xenograft model, P40391942 showed the best antitumor activity, followed by P40621942 > P40531942 > P40751942. Two-six mice treated with P40391942 showed a complete response. [Table 2]

[0300] Because the protease-cleavable linker in P41222037 is easier to cleave than the linker in P43492037, Figures 12A-12B and Table 3 show that P41222037 exhibited better antitumor activity than that of P43492037 in the A375-PBMC xenograft model. [Table 3-1] [Table 3-2]

[0301] Figures 13A-13B and Table 4 show that in the HT-29-PBMC xenograft model, P41222037 demonstrated the best antitumor activity, followed by P43492037 > P42412037 (anti-PD-1 antibody). Three-six mice treated with P41222037 demonstrated complete responses. [Table 4]

[0302] Example 5 In vivo efficacy of different targeting IgG-proIL-2v in the A375-PBMC xenograft model PD-L1-proIL-2v, PD-1-proIL-2v and B7H3-proIL-2v were tested alone for their anti-tumor efficacy in the A375-PBMC xenograft model.

[0303] A375 cells were maintained in vitro in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air. Human PBMCs were cocultured with mitomycin C-treated A375 tumor cells for 6 days and maintained in vitro as suspensions cultured in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air.

[0304] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8–10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark daily cycle. Mice were maintained in individual ventilated cages at a constant temperature (20–26°C) and humidity (40–70%) with ≤6 animals per cage. Animals had free access to radiation-sterilized dry granular food and sterile drinking water throughout the study period. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva prior to implementation. After arrival, animals were maintained for at least 3 days to acclimate to their new environment. During daily monitoring, animals were checked for any effect of tumor growth on normal behavior, such as mobility, food and water intake (visual observation only), weight gain / loss (weight was measured twice a week), dull eyes / coat, and any other abnormal effects.

[0305] Mice were implanted in the right flank with 4 × 10 cells in 0.2 ml of HBSS (1:1) containing 0.1 ml of Matrigel for tumor development. 6 A375 cells and 4 x 10 5hPBMCs (co-cultured with A375) were inoculated subcutaneously. One week after tumor cell inoculation, mice were injected intravenously with PD-L1-proIL-2v, PD-1-proIL-2v, or B7H3-proIL-2v. Mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 where a and b are the longest and shortest diameters of the tumor, respectively. The tumor volume was then used to calculate TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations: TGI%=(1-(T n -T0) / (V n -V0))×100% T / C%=(T n / T0) / (V n / V0)×100%

[0306] In the formula, T n and V n indicates the tumor volume of the treatment group and the vehicle control group, respectively, on the nth day after the start of treatment. TO and V0 indicate the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0307] Figures 14A-14B and Table 5 show that both activated PD-L1-proIL-2v and PD-1-proIL-2v mediated dose-dependent anti-tumor efficacy in the A375-PBMC xenograft model, and that PD-1-proIL-2v demonstrated superior efficacy compared to PD-L1-proIL-2v at doses of 0.1 mg / kg and 0.3 mg / kg. [Table 5]

[0308] Figures 15A-15B and Table 6 show that PD-1-proIL-2v mediated superior efficacy compared to B7H3-proIL-2v at a dose of 0.3 mg / kg in an A375-PBMC xenograft model. These data indicated that PD-1-proIL-2v was a more potent inhibitor of tumor growth compared to other IgG-proIL-2s targeting PD-L1 or B7H3. [Table 6]

[0309] Example 6 In vivo efficacy of PD-L1-proIL-2v and PD-L1-proIL-2wt in an A375-PBMC xenograft model PD-L1-proIL-2v and PD-L1-proIL-2wt were tested alone for their anti-tumor efficacy in an A375-PBMC xenograft model.

[0310] A375 cells were maintained in vitro in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO in air.

[0311] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8–10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark daily cycle. Mice were maintained in individual ventilated cages at a constant temperature (20–26°C) and humidity (40–70%) with ≤6 animals per cage. Animals had free access to radiation-sterilized dry granular food and sterile drinking water throughout the study period. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva prior to implementation. After arrival, animals were maintained for at least 3 days to acclimate to their new environment. During daily monitoring, animals were checked for any effect of tumor growth on normal behavior, such as mobility, food and water intake (visual observation only), weight gain / loss (weight was measured twice a week), dull eyes / coat, and any other abnormal effects.

[0312] Mice were implanted in the right flank with 4 × 10 cells in 0.2 ml of HBSS (1:1) containing 0.1 ml of Matrigel for tumor development. 6 A375 cells and 4 x 10 5 Mice were inoculated subcutaneously with 1000 mg of hPBMCs. One week after tumor cell inoculation, mice were injected intravenously with PD-L1-proIL-2v or PD-L1-proIL-2wt. Mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 where a and b are the longest and shortest diameters of the tumor, respectively. The tumor volume was then used to calculate TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations: TGI%=(1-(T n -T0) / (V n -V0))×100% T / C%=(Tn / T0) / (V n / V0)×100%

[0313] In the formula, T n and V n indicates the tumor volume of the treatment group and the vehicle control group, respectively, on the nth day after the start of treatment. TO and V0 indicate the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0314] Figures 16A-16B and Table 7 show that both PD-L1-proIL-2v and PD-L1-proIL-2wt mediated anti-tumor efficacy in the A375-PBMC xenograft model, and that PD-L1-proIL-2wt demonstrated superior efficacy compared to PD-L1-proIL-2v at a dose of 0.3 mg / kg. [Table 7]

[0315] Example 7 In vivo efficacy of PD-1-proIL-2v and anti-PD-L1 antibodies as single agents and in combination in an A375-PBMC xenograft model PD-1-proIL-2v and anti-PD-L1 antibodies were tested for their anti-tumor efficacy compared to their combination in an A375-PBMC xenograft model.

[0316] A375 cells were maintained in vitro in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air. Human PBMCs were cocultured with mitomycin C-treated A375 tumor cells for 6 days and maintained in vitro as suspensions cultured in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air.

[0317] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8–10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark daily cycle. Mice were maintained in individual ventilated cages at a constant temperature (20–26°C) and humidity (40–70%) with ≤6 animals per cage. Animals had free access to radiation-sterilized dry granular food and sterile drinking water throughout the study period. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva prior to implementation. After arrival, animals were maintained for at least 3 days to acclimate to their new environment. During daily monitoring, animals were checked for any effect of tumor growth on normal behavior, such as mobility, food and water intake (visual observation only), weight gain / loss (weight was measured twice a week), dull eyes / coat, and any other abnormal effects.

[0318] Mice were implanted in the right flank with 4 × 10 cells in 0.2 ml of HBSS (1:1) containing 0.1 ml of Matrigel for tumor development. 6 A375 cells and 4 x 10 5 of hPBMCs (co-cultured with A375) were inoculated subcutaneously. One week after tumor cell inoculation, mice were injected intravenously with PD-1-proIL-2v, anti-PD-L1 antibody, or a combination of PD-1-proIL-2v + PD-L1. Mice in the vehicle group were injected with PBS. Tumor volume was measured in two dimensions using calipers, and the volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 where a and b are the longest and shortest diameters of the tumor, respectively. The tumor volume was then used to calculate TGI and T / C values. Tumor growth inhibition (TGI%) and relative tumor growth inhibition (T / C%) were calculated according to the following equations: TGI%=(1-(T n -T0) / (Vn -V0))×100% T / C%=(T n / T0) / (V n / V0)×100%

[0319] In the formula, T n and V n indicates the tumor volume of the treatment group and the vehicle control group, respectively, on the nth day after the start of treatment. TO and V0 indicate the tumor volume of the corresponding group on the day of grouping. The results were analyzed using Prism GraphPad.

[0320] Figures 17A-17B and Table 8 show that the combination of PD-1-proIL-2v and PD-L1 mediated superior efficacy compared to either monotherapy in the A375-PBMC xenograft model, and that all tumors in the combination group had a complete response. [Table 8]

[0321] Example 8 Pharmacokinetic and pharmacodynamic studies of PD-1-proIL-2v PD-1-proIL-2v was tested for pharmacokinetics and pharmacodynamics in peripheral blood and tumors.

[0322] A375 cells were maintained in vitro in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air. Human PBMCs were cocultured with mitomycin C-treated A375 tumor cells for 6 days and maintained in vitro as suspensions cultured in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO2 in air.

[0323] Female NCG mice (GemPharmatech Co., Ltd., Nanjing, China), 8–10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark daily cycle. Mice were maintained in individual ventilated cages at a constant temperature (20–26°C) and humidity (40–70%) with ≤6 animals per cage. Animals had free access to radiation-sterilized dry granular food and sterile drinking water throughout the study period. All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Viva prior to implementation. After arrival, animals were maintained for at least 3 days to acclimate to their new environment. During daily monitoring, animals were checked for any effect of tumor growth on normal behavior, such as mobility, food and water intake (visual observation only), weight gain / loss (weight was measured twice a week), dull eyes / coat, and any other abnormal effects.

[0324] For pharmacokinetic studies, mice were inoculated with 4 × 10 IgG in 0.2 ml of HBSS (1:1) containing 0.1 ml of Matrigel into the right flank for tumor development. 6 A375 cells and 4 x 10 5 hPBMCs were subcutaneously inoculated. 18 days after tumor cell inoculation, mice were intravenously injected with 1 mg / kg of PD-1-proIL-2v. Blood and tumor samples were collected at 1 hour, 6 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, and 168 hours after dosing. Blood samples were centrifuged within 30 minutes of collection to obtain serum samples (6000 rpm, 5 minutes, 4°C). Serum samples were stored at -80°C before use. Tumors were weighed, and then PBS was added in a volume twice the tumor weight, and the tissue was homogenized on ice. The homogenate was then centrifuged at 6000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored at -80°C before use.

[0325] Total drug (including intact and cleaved procytokines) and procytokines (intact procytokines) were determined using ELISAs employing PD-1 as a capture antibody and various detection antibodies. HRP-conjugated anti-human IgG or anti-IL-2Rα were used to detect total and procytokine levels, respectively. The level of cleaved cytokines (activated cytokines) was calculated by subtracting procytokines from the total drug concentration.

[0326] Figure 18A and Table 9 show that the serum concentrations of P41222037 total drug and procytokine were almost the same, and there were no significant differences in the pharmacokinetic parameters of serum total drug and procytokine, indicating that P41222037 was very stable in peripheral blood. Figures 18B-18C show that the intratumoral concentrations of P41222037 total drug and procytokine peaked at 6 hours, and the intratumoral concentration of procytokine was lower than the intratumoral concentration of total drug at 48 hours (Figure 18B), indicating that P41222037 was cleaved in the tumor to release activated cytokines (18C). [Table 9-1] [Table 9-2]

[0327] For pharmacodynamic studies in peripheral blood, mice were dosed with 5 × 10 6 10 hPBMC cells were injected i.v. 14 days later, 4 × 10 6 A375 cells were subcutaneously inoculated into the right flank of mice for tumor development. One week after tumor cell inoculation, mice were intravenously injected with 1 mg / kg of PD-1-proIL-2v. Mice in the vehicle group were injected with PBS.

[0328] Anticoagulated blood was collected at 0, 72, 168, 240, and 336 hours and lysed with red blood cell lysis buffer. Cells were stained for 30 minutes at 4°C using staining buffer containing CD45 (clone HI30, BioLegend), CD3 (clone UCHT1, BioLegend), CD4 (clone RPA-T4, BD Pharmingen), and CD8 (clone HIT8a, BioLegend). Samples were stained for CD45 (clone HI30, BioLegend), CD3 (clone UCHT1, BioLegend), CD4 (clone RPA-T4, BD Pharmingen), and CD8 (clone HIT8a, BioLegend). + CD3 + cells (CD3 T cells), CD45 + CD3 + CD4 + cells (CD4 T cells) and CD45 + CD3 + CD8 + The cells were analyzed using a flow cytometer (CytoFLEX S, Beckman Coulter) gating on CD8 T cells.

[0329] Figures 19A-19C show CD3 + , CD4 + and CD8 + It shows that T cells did not have a significant difference or increase over time in peripheral blood.

[0330] For pharmacodynamic studies in tumors, mice were inoculated with 4 × 10 IgG in 0.2 ml of HBSS (1:1) containing 0.1 ml of Matrigel in the right flank for tumor development. 6 A375 cells and 4 x 10 4 hPBMCs (co-cultured with A375) were subcutaneously inoculated. 18 days after tumor cell inoculation, mice were intravenously injected with 1 mg / kg of PD-1-proIL-2v. Mice in the vehicle group were injected with PBS.

[0331] On day 12, tumors were harvested, weighed, and dissociated using a validated tumor dissociation kit (Miltenyi Biotec) according to the protocol. The cell suspension was then filtered through a 70 μm cell strainer. Tumor cells were stained with Live / Dead Horizon Fixable Viability Stain 700 (BD Pharmingen) in PBS for 15 minutes. The cells were then stained with staining buffer containing CD45 (clone HI30, BioLegend), CD3 (clone UCHT1, BioLegend), CD4 (clone RPA-T4, BD Pharmingen), and CD8 (clone HIT8a, BioLegend) for 30 minutes at 4°C. Samples were analyzed for CD45. + CD3 + cells (CD3 T cells), CD45 + CD3 + CD4 + cells (CD4 T cells) and CD45 + CD3 + CD8 + The cells were analyzed using a flow cytometer (CytoFLEX S, Beckman Coulter) gating on CD8 T cells.

[0332] Figures 20A-20C show CD3 + , CD4 + and CD8 + All show that T cells are increased at the tumor site.

[0333] In summary, these data demonstrate that the PD-1-proIL-2v fusion protein P41222037 is highly stable, releases little activated cytokines into the peripheral blood (reducing toxicity), and can be cleaved in the tumor, releasing activated cytokines to activate CD3 + , CD4 + and CD8 + They show that it stimulates T cell proliferation, which reduces toxicity in the peripheral blood and selectively enhances antitumor activity in the TME.

[0334] Example 9 Biological activity of PD-1-proIL-2v in cynomolgus monkeys The in vivo bioactivity of masked and unmasked PD-1-proIL-2v was evaluated in vivo in cynomolgus monkeys.

[0335] A dose-ranging study was conducted to examine the safety profile and pharmacodynamics of PD-1-proIL-2v (P41222037) in cynomolgus monkeys. Each group, containing one male and one female cynomolgus monkey, received five repeat doses of 3, 10, and 30 mg / kg P41222037 (masked PD-1-proIL-2v) or 1 mg / kg P41252037 (unmasked PD-1-proIL-2v).

[0336] Blood samples for pharmacodynamic studies were collected pre-dose and 5 minutes, 1 hour, 6 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, and 168 hours after the first dose. P41222037 concentrations were determined using an ELISA with PD-1 as the capture antibody and anti-IL-2Rα as the detection antibody. P41252037 concentrations were determined using an ELISA with PD-1 as the capture antibody and HRP-conjugated anti-human IgG as the detection antibody.

[0337] Figure 21A shows that serum concentrations of P41222037 in cynomolgus monkeys demonstrated dose-dependent procytokine exposure, as well as T of masked PD-1-proIL-2v (P41222037) compared to unmasked PD-1-proIL-2v (P41222037). 1 / 2 This indicates that it was long.

[0338] Blood samples for serum chemistry were collected before dosing and 4, 11, 18, 25, and 30 days after dosing. Changes in albumin were monitored using an automated hematology analyzer. Figure 21B shows that there was no significant decrease in albumin levels in cynomolgus monkeys treated with 3, 10, and 30 mg / kg P41222037 compared to vehicle control. All monkeys tolerated all three doses of P41222037 well.

[0339] Example 10 Effect of linker length and modification on immune cell stimulation Experiments were performed to examine the effect of stable linker length (between Fc and IL-2) on cell proliferation and on the activity of the proprotein and activated protein toward specific immune cell subsets. Constructs tested included P41222037 (4 amino acid stable linker), P45412037 (8 amino acid stable linker), and P45422037 (12 amino acid stable linker) with and without MMP-2 protease treatment compared to human IL-2. Figure 26 shows the heavy chain / IL-2 junction of the constructs tested.

[0340] Proliferation of M-07e cells by PD1-proIL-2v. Human M-07e cells (expressing human IL-2Rβγ) were cultured in RPMI 1640 supplemented with 20% fetal bovine serum (FBS) and 10% 5637 cell culture supernatant. To measure cytokine-dependent cell proliferation, M-07e cells were harvested in their logarithmic growth phase and washed twice with PBS. 90 μl of cell suspension (2 × 10 4Cells (1000 cells / well) were seeded into 96-well plates and incubated in assay medium for cytokine starvation (RPMI 1640 supplemented with 10% FBS) at 37°C and 5% CO2 for 4 hours. IL-2, PD1-proIL-2v (P41222037, P45412037, and P45422037), or MMP-2-cleaved PD1-proIL-2v protein samples used in the assay were prepared in assay medium to initial concentrations (300 nM for IL-2, 8100 nM for PD1-proIL-2v, and 2700 nM for MMP-2-activated PD1-proIL-2v fusion protein) and then serially diluted 1 / 3. 10 μl of diluted protein was added to the corresponding wells and incubated at 37°C and 5% CO2 for 72 hours. A colorimetric assay using Cell Counting Kit-8 (CCK-8, Dojindo, CK04) was performed to measure the amount of viable cells.

[0341] As shown in Figure 22A, the P41222037 proprotein failed to induce proliferation of M-07e cells at the highest concentration tested; MMP-2 cleavage restored the biological activity of P41222037, but the activity was approximately 30-fold lower than that of wild-type IL-2. As shown in Figures 22B-22C, the P45412037 and P45422037 proproteins had no activity at the highest concentration of 810 nM; MMP-2 cleavage restored the activity of P45412037 or P45422037, but the activity was approximately 34-fold or 25-fold lower than that of wild-type IL-2, respectively.

[0342] STAT5 phosphorylation upon treatment of resting human PBMCs with PD-1-proIL2. Frozen human PBMCs (SAILYBIO, donor 1) were allowed to recover in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin for approximately 2 hours at 37°C in a 5% CO atmosphere. PBMC cells were then cultured at 2.6 x 10 6Adjust the cells / ml and incubate at 4 x 10 cells per well in a 96-well plate. 5 Recovered PBMCs were incubated with rhIL-2, P41222037, PD-1-proIL-2v linker length variants, or MMP-2-activated proteins for 15 minutes at 37°C.

[0343] After incubation, cells were immediately fixed with Cytofix buffer (BD Bioscience) and stored on ice for 15 minutes to determine phosphorylation status. Then, they were washed once with BD Pharmingen™ staining buffer (FBS). For surface staining, cells were incubated with CD3 Alexa Flour 700 (BD 557943), CD4 PerCP-Cytm5.5 (BD 560650), CD8 APC-Cytm7 (BD 557760), CD25 BV421 (BD 562442), and CD56 BV510 (BD 744218) for 30 minutes at 4°C. Then, they were washed once with 1x PBS, centrifuged at 500g for 5 minutes, and the supernatant was removed by aspiration. Then, cells were permeabilized with pre-chilled Phosflow Perm Buffer III (BD Bioscience) for 30 minutes at 4°C.

[0344] Before starting intracellular staining, cells were washed once with 1x PBS, centrifuged at 500g for 5 minutes, and the cell pellet was collected. Then, cells were stained with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa Fluor® 647 (BD 562076) at room temperature for 40 minutes. Cells were washed twice with BD Pharmingen™ staining buffer (FBS) and centrifuged at 500g for 5 minutes to pellet the cells. Each cell pellet was resuspended in 200ul (per well) of BD Pharmingen staining buffer (FBS), and data were acquired using flow cytometry. STAT5 phosphorylation status in PBMC subsets during prodrug treatment was acquired and processed using CytoFLEX (Beckman).

[0345] Figure 23A shows STAT phosphorylation in CD4 T cells, CD8 T cells, and regulatory T cells upon treatment of resting PBMCs from donor 1 with rhIL-2 and intact and activated P41222037. Concentrations as high as 1000 nM of intact P41222037 proprotein failed to induce STAT5 phosphorylation in all cell subsets tested. The protease-activated form of P41222037 (MMP-2 cleaved) was equally effective in inducing STAT5 phosphorylation in CD8 and CD4 T cells. However, in Treg cells, the activated form of P41222037 was approximately 30-fold less potent than huIL-2. Figures 23B-23C show STAT5 phosphorylation in CD4 T cells, CD8 T cells, and Tregs upon treatment of resting PBMCs with intact and MMP2-cleaved P45412037 or P45422037 compared to human recombinant IL-2. Figure 23D shows that, unlike P41222037, both intact P45412037 and P45422037 induced STAT5 phosphorylation in CD4 T and regulatory T cells, but not in CD8 T cells.

[0346] Figures 24A-24C show the activity of additional proprotein constructs (see Figure 26) in this assay. Compared to human recombinant IL-2, the intact P78192037 and P78342037 proproteins failed to induce STAT5 phosphorylation in all cell subsets tested. Surprisingly, the intact P78362037 proprotein had significant activity on STAT5 phosphorylation in CD4 T and regulatory T cells, but not in CD8 T cells; the intact P78352037 proprotein had weaker activity on STAT5 phosphorylation in CD4 T and regulatory T cells. The intact proproteins of P78192037, P78202037, P78212037, P78222037, P78232037, P78242037, P78252037, P78262037, P78272037, P78282037, P78292037, P78302037, P78312037, P78322037, P78332037, and P78342037 showed no activity on STAT5 phosphorylation in CD4 T cells and regulatory T cells.

[0347] STAT5 phosphorylation upon treatment of pre-activated human PBMCs with PD-1-proIL2. Frozen human PBMCs (SAILYBIO, donor 1) were recovered in RPMI-1640 supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin for 2 hours at 37°C in a 5% CO atmosphere. For assays performed on pre-activated PBMCs, 10 cm plates were coated overnight at 4°C with αCD3 (1 μg / ml, BioLegend). PBMCs suspended in RPMI-1640 supplemented with soluble αCD28 (1 μg / ml, BioLegend) were cultured for 3 days at 37°C in a 5% CO atmosphere. Pre-activated PBMCs were cultured at 2.6 × 10 6 Adjust the cell density to 4 x 10 cells / ml 5 Cells / well were plated in 96-well plates.

[0348] After incubation with rhIL-2 or P41222037 for 15 minutes at 37°C, cells were immediately fixed with Cytofix buffer (BD Bioscience), stored on ice for 15 minutes, and washed once with BD Pharmingen™ staining buffer (FBS). For surface staining, cells were incubated with CD3 Alexa Flour 700 (BD 557943), CD4 PerCP-Cytm5.5 (BD 560650), CD8 APC-Cytm7 (BD 557760), and CD25 BV421 (BD 562442) for 30 minutes at 4°C. Cells were washed once with 1x PBS and centrifuged at 500g for 5 minutes. Cells were permeabilized with pre-chilled Phosflow Perm buffer III (BD Bioscience) for 30 minutes at 4°C.

[0349] Prior to intracellular staining, cells were washed once with 1x PBS and centrifuged at 500g for 5 minutes. Then, cells were stained with Foxp3 PE (BD 560046) and anti-Stat5 (pY694) Alexa Fluor® 647 (BD 562076) for 40 minutes at RT. Cells were washed twice with BD Pharmingen™ staining buffer (FBS) and centrifuged at 500g for 5 minutes. Finally, each cell pellet was resuspended in 200ul (per well) of BD Pharmingen staining buffer (FBS) for FACS data acquisition using flow cytometry. STAT5 phosphorylation status in PBMC subsets during prodrug treatment was acquired and processed by CytoFLEX (Beckman).

[0350] Figure 25 shows STAT phosphorylation in CD4 T cells, CD8 T cells, and Treg cells upon treatment of pre-activated PBMCs from donor 1 with rhIL-2 or P41222037. Up to 300 nM of intact P41222037 proprotein did not induce STAT5 phosphorylation in all cell subsets tested.

[0351] Overall, constructs such as the P41222037 proprotein with a shorter stable linker (e.g., 7 amino acids or less) between the Fc and IL-2 may be able to inhibit the immunosuppressive CD4 T reg , and do not stimulate certain other immune cells, and are therefore unexpectedly useful compared to constructs with longer stable linkers (e.g., 8 amino acids or longer) for treating diseases such as cancer that benefit from highly regulated activation of anti-tumor immune cell responses.

Claims

1. An activatable proprotein homodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are oriented from the N-terminus to the C-terminus. It specifically binds to human PD-1 and comprises a heavy chain variable (VH) region including the VHCDR1, VHCDR2, and VHCDR3 regions shown in SEQ ID NO: 3; and an antigen-binding fragment (Fab) region including the light chain variable (VL) region including the VLCDR1, VLCDR2, and VLCDR3 regions shown in SEQ ID NO: 4, a hinge / Fc domain, a first linker, an IL-2 protein, a second linker, and an IL-2Rα protein. The hinge / Fc domain of the first polypeptide binds to the hinge / Fc domain of the second polypeptide, the IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, the IL-2Rα of the first polypeptide binds to the IL-2 protein of the second polypeptide, and the binding masks the binding site of the IL-2 protein(s) that otherwise binds to the IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo. The second linker is a cleavable linker, the first and second polypeptides contain an amino acid sequence that is at least 95% identical to SEQ ID NO: 146, and the proprotein contains an anti-PD-1 light chain that is at least 95% identical to SEQ ID NO:

147. An activatable proprotein homodimer.

2. The activatable proprotein homodimer according to claim 1, wherein the VH region includes SEQ ID NO: 3 and the VL region includes SEQ ID NO:

4.

3. The activatable proprotein homodimer according to claim 1, wherein the hinge comprises Sequence ID No. 42, and the Fc domain is a modified IgG1 CH2 domain having an L234A / L235A ("LALA") mutation and / or a P329A or P329G mutation (EU numbered).

4. The activatable proprotein homodimer according to claim 1, wherein the IL-2 protein comprises SEQ ID NO: 84 or 85.

5. The activatable proprotein homodimer according to claim 1, wherein the IL-2Rα protein comprises SEQ ID NO:

90.

6. The activatable proprotein homodimer according to claim 1, wherein the first linker is a stable four-amino acid linker GGGS (SEQ ID NO: 188), and the cleavable linker includes the protease cleavage site of SEQ ID NO:

93.

7. The activatable proprotein homodimer according to claim 1, wherein the Fab comprises SEQ ID NO: 3 (VH) and the human IgG1 CH1 domain and SEQ ID NO: 4 (VL) and the CL domain (human kappa); the Fc domain comprises the IgG1 hinge of SEQ ID NO: 42, the modified human IgG1 CH2 domain of SEQ ID NO: 57, and the human IgG1 CH3 domain of SEQ ID NO: 58; the first linker is the 4-amino acid stable linker of SEQ ID NO: 188; the IL-2 protein comprises SEQ ID NO: 84 or 85 having R38D, K43E, and C125S mutations as desired; the second linker is the protease-cleavable linker of SEQ ID NO: 93; and the IL-2Rα protein comprises SEQ ID NO: 88 or 90 having D6R and E29K mutations as desired.

8. The activatable proprotein homodimer according to claim 1, wherein the first polypeptide and the second polypeptide comprise SEQ ID NO: 146, and the proprotein comprises the anti-PD-1 light chain polypeptide of SEQ ID NO:

147.

9. One or more recombinant nucleic acid molecules encoding an activatable proprotein homodimer according to any one of claims 1 to 8.

10. One or more recombinant nucleic acid molecules according to claim 9, wherein the first recombinant nucleic acid molecule encodes the VH / CH1 region of the Fab region, the hinge / Fc domain, the first linker, the IL-2 protein, the second linker, and the IL-2Rα protein, and the second nucleic acid molecule encodes the anti-PD-1 light chain.

11. One or more vectors comprising one or more recombinant nucleic acid molecules as described in claim 9.

12. A host cell comprising one or more recombinant nucleic acid molecules according to claim 9, or one or more vectors comprising one or more recombinant nucleic acid molecules according to claim 9.

13. A method for producing an activatable proprotein, comprising the steps of: culturing the host cells described in claim 12 under culture conditions suitable for the expression of the activatable proprotein homodimer; and isolating the activatable proprotein from the culture.

14. A pharmaceutical composition comprising an activatable proprotein homodimer according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition according to claim 14 for use in the treatment of a disease in a subject, wherein the disease is cancer.

16. The pharmaceutical composition for use according to claim 15, wherein the cancer expresses or overexpresses PD-L1.

17. The pharmaceutical composition for use according to claim 15, wherein the cancer is primary or metastatic cancer, and is selected from one or more of the following: melanoma (or metastatic melanoma, if necessary), kidney cancer (or renal cell carcinoma, if necessary), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (or lymphocytic leukemia, chronic myeloid leukemia, acute myelocytic leukemia, or relapsed acute myelocytic leukemia, if necessary), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, undifferentiated neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

18. The pharmaceutical composition for use according to claim 15, wherein, after administration, the activatable proprotein homodimer is activated via protease cleavage, which exposes the binding site(s) of the IL-2 protein that binds in vitro or in vivo to the IL-2Rβ / γc chain present on the surface of the immune cells in cancer cells or cancer tissue or the tumor microenvironment (TME), thereby generating an activated protein.

19. Use of the pharmaceutical composition according to claim 14 in the manufacture of a pharmaceutical for treating cancer.

20. The use according to claim 19, wherein the cancer expresses or overexpresses PD-L1.

21. The use according to claim 19, wherein the cancer is primary cancer or metastatic cancer, and is selected from one or more of the following: melanoma (if necessary, metastatic melanoma), kidney cancer (if necessary, renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (if necessary, lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, undifferentiated neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

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